Methods and Compositions for the Treatment of Gastrointestinal Disorders

ABSTRACT

The present invention features compositions and related methods for treating IBS and other gastrointestinal disorders and conditions (e.g., gastrointestinal motility disorders, functional gastrointestinal disorders, gastroesophageal reflux disease (GERD), Crohn&#39;s disease, ulcerative colitis, Inflammatory bowel disease, functional heartburn, dyspepsia (including functional dyspepsia or nonulcer dyspepsia), gastroparesis, chronic intestinal pseudo-obstruction (or colonic pseudo-obstruction), and disorders and conditions associated with constipation, e.g., constipation associated with use of opiate pain killers, post-surgical constipation (post-operative ileus), and constipation associated with neuropathic disorders as well as other conditions and disorders using peptides and other agents that activate the guanylate cyclase C (GC-C) receptor.

CLAIM OF PRIORITY

This application is a divisional of U.S. Utility patent application Ser.No. 11/949,340, filed Dec. 3, 2007, which claims priority to U.S.Utility patent application Ser. No. 10/796,719, filed Mar. 9, 2004,which claims priority to U.S. Utility patent application Ser. No.10/766,735, filed Jan. 28, 2004, which claims priority under 35 USC§119(e) to U.S. Provisional Patent Application Ser. No. 60/443,098,filed on Jan. 28, 2003; U.S. Provisional Patent Application Ser. No.60/471,288, filed on May 15, 2003 and U.S. Provisional PatentApplication Ser. No. 60/519,460, filed on Nov. 12, 2003, the entirecontents of all of these priority applications are hereby incorporatedby reference.

TECHNICAL FIELD

This invention relates to methods and compositions for treating variousdisorders, including gastrointestinal disorders, obesity, congestiveheart failure and benign prostatic hyperplasia.

BACKGROUND

Irritable bowel syndrome (IBS) is a common chronic disorder of theintestine that affects 20 to 60 million individuals in the US alone(Lehman Brothers, Global Healthcare-Irritable bowel syndrome industryupdate, September 1999). IBS is the most common disorder diagnosed bygastroenterologists (28% of patients examined) and accounts for 12% ofvisits to primary care physicians (Camilleri 2001, Gastroenterology120:652-668). In the US, the economic impact of IBS is estimated at $25billion annually, through direct costs of health care use and indirectcosts of absenteeism from work (Talley 1995, Gastroenterology109:1736-1741). Patients with IBS have three times more absenteeism fromwork and report a reduced quality of life. Sufferers may be unable orunwilling to attend social events, maintain employment, or travel evenshort distances (Drossman 1993, Dig Dis Sci 38:1569-1580). There is atremendous unmet medical need in this population since few prescriptionoptions exist to treat IBS.

Patients with IBS suffer from abdominal pain and a disturbed bowelpattern. Three subgroups of IBS patients have been defined based on thepredominant bowel habit: constipation-predominant (c-IBS),diarrhea-predominant (d-IBS) or alternating between the two (a-IBS).Estimates of individuals who suffer from c-IBS range from 20-50% of theIBS patients with 30% frequently cited. In contrast to the other twosubgroups that have a similar gender ratio, c-IBS is more common inwomen (ratio of 3:1) (Talley et al. 1995, Am J Epidemiol 142:76-83).

The definition and diagnostic criteria for IBS have been formalized inthe “Rome Criteria” (Drossman et al. 1999, Gut 45:Suppl II: 1-81), whichare well accepted in clinical practice. However, the complexity ofsymptoms has not been explained by anatomical abnormalities or metabolicchanges. This has led to the classification of IBS as a functional GIdisorder, which is diagnosed on the basis of the Rome criteria andlimited evaluation to exclude organic disease (Ringel et al. 2001, AnnuRev Med 52: 319-338). IBS is considered to be a “biopsychosocial”disorder resulting from a combination of three interacting mechanisms:altered bowel motility, an increased sensitivity of the intestine orcolon to pain stimuli (visceral sensitivity) and psychosocial factors(Camilleri 2001, Gastroenterology 120:652-668). Recently, there has beenincreasing evidence for a role of inflammation in etiology of IBS.Reports indicate that subsets of IBS patients have small but significantincreases in colonic inflammatory and mast cells, increased induciblenitric oxide (NO) and synthase (iNOS) and altered expression ofinflammatory cytokines (reviewed by Talley 2000, Medscape Coverage ofDDW week).

SUMMARY

The present invention features compositions and related methods fortreating IBS and other gastrointestinal disorders and conditions (e.g.,gastrointestinal motility disorders, functional gastrointestinaldisorders, gastroesophageal reflux disease (GERD), Crohn's disease,ulcerative colitis, Inflammatory bowel disease, functional heartburn,dyspepsia (including functional dyspepsia or nonulcer dyspepsia),gastroparesis, chronic intestinal pseudo-obstruction (or colonicpseudo-obstruction), and disorders and conditions associated withconstipation, e.g., constipation associated with use of opiate painkillers, post-surgical constipation, and constipation associated withneuropathic disorders as well as other conditions and disorders. Thecompositions feature peptides that activate the guanylate cyclase C(GC-C) receptor.

The present invention also features compositions and related methods fortreating obesity, congestive heart failure and benign prostatichyperplasia (BPH).

Without being bound by any particular theory, in the case of IBS andother gastrointestinal disorders the peptides are useful because theycan increase gastrointestinal motility.

Without being bound by any particular theory, in the case of IBS andother gastrointestinal disorders the peptides are useful, in part,because they can decrease inflammation.

Without being bound by any particular theory, in the case of IBS andother gastrointestinal disorders the peptides are also useful becausethey can decrease gastrointestinal pain or visceral pain.

The invention features pharmaceutical compositions comprising certainpeptides that are capable of activating the guanylate-cyclase C (GC-C)receptor. Also within the invention are pharmaceutical compositionscomprising a peptide of the invention as well as combinationcompositions comprising a peptide of the invention and a secondtherapeutic agent, e.g., an agent for treating constipation (e.g., achloride channel activator such as SPI-0211; Sucampo Pharmaceuticals,Inc.; Bethesda, Md., a laxative such as MiraLax; Braintree Laboratories,Braintree Mass.) or some other gastrointestinal disorder. Examples of asecond therapeutic agent include: acid reducing agents such as protonpump inhibitors (e.g. omeprazole, esomeprazole, lansoprazole,pantorazole and rabeprazole) and H2 receptor blockers (e.g. cimetidine,ranitidine, famotidine and nizatidine), pro-motility agents such asmotilin agonists (e.g. GM-611 or mitemcinal fumarate), and 5HT receptoragonists (e.g. 5HT4 receptor agonists such as Zelnorm®; 5HT3 receptoragonists such as MKC-733), 5HT receptor antagonists (e.g. 5HT1, 5HT2,5HT3 (e.g. alosetron), and 5HT4 receptor antagonists; muscarinicreceptor agonists, anti-inflammatory agents, antispasmodics,antidepressants, centrally-acting analgesic agents such as opiodreceptor agonists, opiod receptor antagonists (e.g. naltrexone), agentsfor the treatment of Inflammatory bowel disease, Crohn's disease andulcerative colitis (e.g., Traficet-EN™ (ChemoCentryx, Inc.; San Carlos,Calif.) agents that treat gastrointestinal or visceral pain and cGMPphosphodiesterase inhibitors (motapizone, zaprinast, and suldinacsulfone). The peptides of the invention can also be used in combinationwith agents such a tianeptine (Stablon®) and other agents described inU.S. Pat. No. 6,683,072; (E)-4(1,3bis(cyclohexylmethyl)-1,2,34,-tetrahydro-2,6-diono-9H-purin-8-yl)cinnamicacid nonaethylene glycol methyl ether ester and related compoundsdescribed in WO 02/067942. The peptides can also be used in combinationwith treatments entailing the administration of microorganisms useful inthe treatment of gastrointestinal disorders such as IBS. Probactrix®(The BioBalance Corporation; New York, N.Y.) is one example of aformulation that contains microorganisms useful in the treatment ofgastrointestinal disorders. In addition, the pharmaceutical compositionscan include an (OK) agent selected from the group consisting of: Cachannel blockers (e.g., ziconotide), 5HT receptor agonists (e.g. 5HT1,5HT2, 5HT3 and 5HT4 receptor agonists) 5HT receptor antagonists (e.g.5HT1, 5HT2, 5HT3 and 5HT4), opioid receptor agonists (e.g., loperamide,fedotozine, and fentanyl, naloxone, naltrexone, methyl nalozone,nalmefene, cypridime, beta funaltrexamine, naloxonazine, naltrindole,and nor-binaltorphimine, morphine, diphenyloxylate, enkephalinpentapeptide, and trimebutine), NK1 receptor antagonists (e.g.,ezlopitant and SR-14033, SSR-241585), CCK receptor agonists (e.g.,loxiglumide), NK1 receptor antagonists, NK3 receptor antagonists (e.g.,talnetant, osanetant SR-142801, SSR-241585), norepinephrine-serotoninreuptake inhibitors (NSRI; e.g., milnacipran), vanilloid and cannabanoidreceptor agonists (e.g., arvanil), sialorphin, sialorphin-relatedpeptides comprising the amino acid sequence QHNPR (SEQ ID NO:111) forexample, VQHNPR (SEQ ID NO:112); VRQHNPR (SEQ ID NO:113); VRGQHNPR (SEQID NO:114); VRGPQHNPR (SEQ ID NO:115); VRGPRQHNPR (SEQ ID NO:116);VRGPRRQHNPR (SEQ ID NO:117); and RQHNPR (SEQ ID NO:118), compounds orpeptides that are inhibitors of neprilysin, frakefamide(H-Tyr-D-Ala-Phe(F)-Phe-NH₂; WO 01/019849 A1), loperamide, Tyr-Arg(kyotorphin), CCK receptor agonists (caerulein), conotoxin peptides,peptide analogs of thymulin, loxiglumide, dexloxiglumide (the R-isomerof loxiglumide) (WO 88105774) and other analgesic peptides or compoundscan be used with or linked to the peptides of the invention.

The invention includes methods for treating various gastrointestinaldisorders by administering a peptide that acts as a partial or completeagonist of the GC-C receptor. The peptide includes at least sixcysteines that form three disulfide bonds. In certain embodiments thedisulfide bonds are replaced by other covalent cross-links and in somecases the cysteines are substituted by other residues to provide foralternative covalent cross-links. The peptides may also include at leastone trypsin or chymotrypsin cleavage site and/or a carboxy-terminalanalgesic peptide or small molecule, e.g., AspPhe or some otheranalgesic peptide. When present within the peptide, the analgesicpeptide or small molecule may be preceded by a chymotrypsin or trypsincleavage site that allows release of the analgesic peptide or smallmolecule. The peptides and methods of the invention are also useful fortreating pain and inflammation associated with various disorders,including gastrointestinal disorders. Certain peptides include afunctional chymotrypsin or trypsin cleavage site located so as to allowinactivation of the peptide upon cleavage. Certain peptides having afunctional cleavage site undergo cleavage and gradual inactivation inthe digestive tract, and this is desirable in some circumstances. Incertain peptides, a functional chymotrypsin site is altered, increasingthe stability of the peptide in vivo.

The invention includes methods for treating other disorders such ascongestive heart failure and benign prostatic hyperplasia byadministering a peptide or small molecule (parenterally or orally) thatacts as an agonist of the GC-C receptor. Such agents can be used incombination with natriuretic peptides (e.g., atrial natriuretic peptide,brain natriuretic peptide or C-type natriuretic peptide), a diuretic, oran inhibitor of angiotensin converting enzyme.

The invention features methods and compositions for increasingintestinal motility. Intestinal motility involves spontaneouscoordinated dissentions and contractions of the stomach, intestines,colon and rectum to move food through the gastrointestinal tract duringthe digestive process.

In certain embodiments the peptides include either one or two or morecontiguous negatively charged amino acids (e.g., Asp or Glu) or one ortwo or more contiguous positively charged residues (e.g., Lys or Arg) orone or two or more contiguous positively or negatively charged aminoacids at the carboxy terminus. In these embodiments all of the flankingamino acids at the carboxy terminus are either positively or negativelycharged. In other embodiments the carboxy terminal charged amino acidsare preceded by a Leu. For example, the following amino acid sequencescan be added to the carboxy terminus of the peptide: Asp; Asp Lys; LysLys Lys Lys Lys Lys (SEQ ID NO:123); Asp Lys Lys Lys Lys Lys Lys (SEQ IDNO:124); Leu Lys Lys; and Leu Asp. It is also possible to simply add Leuat the carboxy terminus.

In a first aspect, the invention features a peptide comprising,consisting of, or consisting essentially of the amino acid sequence (I):Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅Cys₆ Cys₇ Xaa₈ Xaa₉ Cys₁₀ Cys₁₁ Xaa₁₂ Xaa₁₃Xaa₁₄ Cys₁₅ Xaa₁₆ Xaa₁₇ Cys₁₈ Xaa₁₉ Xaa₂₀ Xaa₂₁ (SEQ ID NO:119) wherein:Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ is Asn Ser Ser Asn Tyr (SEQ ID NO:121) or ismissing or Xaa₁ Xaa₂ Xaa₃ Xaa₄ is missing. In certain embodiments Xaa₈,Xaa₉, Xaa₁₂, Xaa₁₃, Xaa₁₄, Xaa₁₇, and Xaa₁₉ can be any amino acid. Incertain embodiments Xaa₅ is Asn, Trp, Tyr, Asp, or Phe. In otherembodiments, Xaa₅ can also be Thr or Ile. In other embodiments Xaa₅ isTyr, Asp or Trp. In some embodiments Xaa₈ is Glu, Asp, Gln, Gly or Pro.In other embodiments Xaa₈ is Glu; in some embodiments Xaa₉ is Leu, Ile,Val, Ala, Lys, Arg, Trp, Tyr or Phe in some embodiments Xaa₉ is Leu,Ile, Val, Lys, Arg, Trp, Tyr or Phe.

In certain embodiments, the peptide includes disulfide bonds betweenCys₆ and Cys₁₁, between Cys₇ and Cys₁₅ and between Cys₁₀ and Cys₁₆. Inother embodiments, the peptide is a reduced peptide having no disulfidebonds. In still other embodiments the peptide has one or two disulfidebonds selected from the group consisting of: a disulfide bond betweenCys₆ and Cys₁₁, a disulfide bond between Cys₇ and Cys₁₅ and a disulfidebond between Cys₁₀ and Cys₁₆.

In certain embodiments, an amino acid can be replaced by a non-naturallyoccurring amino acid or a naturally or non-naturally occurring aminoacid analog. For example, an aromatic amino acid can be replaced by3,4-dihydroxy-L-phenylalanine, 3-iodo-L-tyrosine, triiodothyronine,L-thyroxine, phenylglycine (Phg) or nor-tyrosine (norTyr). Phg andnorTyr and other amino acids including Phe and Tyr can be substitutedby, e.g., a halogen, —CH3, —OH, —CH₂NH₃, —C(O)H, —CH₂CH₃, —CN,—CH₂CH₂CH₃, —SH, or another group. Further examples of unnatural aminoacids include: an unnatural analogue of tyrosine; an unnatural analogueof glutamine; an unnatural analogue of phenylalanine; an unnaturalanalogue of serine; an unnatural analogue of threonine; an alkyl, aryl,acyl, azido, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl,alkynl, ether, thiol, sulfonyl, seleno, ester, thioacid, borate,boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine,aldehyde, hydroxylamine, keto, or amino substituted amino acid, or anycombination thereof; an amino acid with a photoactivatable cross-linker;a spin-labeled amino acid; a fluorescent amino acid; an amino acid witha novel functional group; an amino acid that covalently or noncovalentlyinteracts with another molecule; a metal binding amino acid; ametal-containing amino acid; a radioactive amino acid; a photocagedand/or photoisomerizable amino acid; a biotin or biotin-analoguecontaining amino acid; a glycosylated or carbohydrate modified aminoacid; a keto containing amino acid; amino acids comprising polyethyleneglycol or polyether; a heavy atom substituted amino acid (e.g., an aminoacid containing deuterium, tritium, ¹³C, ¹⁵N, or ¹⁸O); a chemicallycleavable or photocleavable amino acid; an amino acid with an elongatedside chain; an amino acid containing a toxic group; a sugar substitutedamino acid, e.g., a sugar substituted serine or the like; acarbon-linked sugar-containing amino acid; a redox-active amino acid; anα.-hydroxy containing acid; an amino thio acid containing amino acid; anα, α disubstituted amino acid; a β-amino acid; a cyclic amino acid otherthan proline; an O-methyl-L-tyrosine; an L-3-(2-naphthyl)alanine; a3-methyl-phenylalanine; a p-acetyl-L-phenylalanine; an0-4-allyl-L-tyrosine; a 4-propyl-L-tyrosine; atri-O-acetyl-GlcNAcβ-serine; an L-Dopa; a fluorinated phenylalanine; anisopropyl-L-phenylalanine; a p-azido-L-phenylalanine; ap-acyl-L-phenylalanine; a p-benzoyl-L-phenylalanine; an L-phosphoserine;a phosphonoserine; a phosphonotyrosine; a p-iodo-phenylalanine; a4-fluorophenylglycine; a p-bromophenylalanine; ap-amino-L-phenylalanine; a isopropyl-L-phenylalanine;L-3-(2-naphthyl)alanine; an amino-, isopropyl-, or O-allyl-containingphenylalanine analogue; a dopa, O-methyl-L-tyrosine; a glycosylatedamino acid; a p-(propargyloxy)phenylalanine, dimethyl-Lysine,hydroxy-proline, mercaptopropionic acid, methyl-lysine,3-nitro-tyrosine, norleucine, pyro-glutamic acid, Z (Carbobenzoxyl),ε-Acetyl-Lysine, β-alanine, aminobenzoyl derivative, aminobutyric acid(Abu), citrulline, aminohexanoic acid, aminoisobutyric acid,cyclohexylalanine, d-cyclohexylalanine, hydroxyproline, nitro-arginine,nitro-phenylalanine, nitro-tyrosine, norvaline, octahydroindolecarboxylate, ornithine, penicillamine, tetrahydroisoquinoline,acetamidomethyl protected amino acids and a pegylated amino acid.Further examples of unnatural amino acids can be found in U.S.20030108885, U.S. 20030082575, and the references cited therein.

Methods to manufacture peptides containing unnatural amino acids can befound in, for example, U.S. 20030108885, U.S. 20030082575, Deiters etal., J Am Chem Soc. (2003) 125:11782-3, Chin et al., Science (2003)301:964-7, and the references cited therein.

The peptides of the invention can be modified using standardmodifications. Modifications may occur at the amino (N-), carboxy (C-)terminus, internally or a combination of any of the preceeding. In oneaspect of the invention, there may be more than one type of modificationon the peptide. Modifications include but are not limited to:acetylation, amidation, biotinylation, cinnamoylation, farnesylation,formylation, myristoylation, palmitoylation, phosphorylation (Ser, Tyror Thr), stearoylation, succinylation, sulfurylation and cyclisation(via disulfide bridges or amide cyclisation), and modification by Cy3 orCy5. The peptides of the invention may also be modified by2,4-dinitrophenyl (DNP), DNP-lysin, modification by7-Amino-4-methyl-coumarin (AMC), flourescein, NBD(7-Nitrobenz-2-Oxa-1,3-Diazole), p-nitro-anilide, rhodamine B, EDANS(5-((2-aminoethyl)amino)naphthalene-1-sulfonic acid), dabcyl, dabsyl,dansyl, texas red, FMOC, and Tamra (Tetramethylrhodamine). The peptidesof the invention may also be conjugated to, for example, BSA or KLH(Keyhole Limpet Hemocyanin).

In some embodiments Xaa₁₂ is Asn, Tyr, Asp or Ala. In other embodimentsXaa₁₂ is Asn. In some embodiments Xaa₁₃ is Ala, Pro or Gly, and in otherembodiments it is Pro. In some embodiments Xaa₁₄ is Ala, Leu, Ser, Gly,Val, Glu, Gln, Ile, Leu, Lys, Arg, or Asp, and in other embodiments itis Ala or Gly, and in still other embodiments it is Ala. In someembodiments Xaa₁₆ is Thr, Ala, Asn, Lys, Arg, Trp; Xaa_(r) is Gly, Proor Ala; Xaa₁₉ is selected from Trp, Tyr, Phe, Asn and Leu or Xaa₁₉ isselected from Trp, Tyr, and Phe or Xaa₁₉ is selected from Leu, Ile andVal; or Xaa₁₉ is His or Xaa₁₉ is selected from Trp, Tyr, Phe, Asn, Ile,Val, His and Leu; and Xaa₂₀ Xaa₂₁ is AspPhe or is missing or Xaa₂₀ isAsn or Glu and Xaa₂₁ is missing or Xaa₁₉ Xaa₂₀ Xaa₂₁ is missing. Theinvention also features methods for treating a gastrointestinal disorder(e.g., a gastrointestinal motility disorder, a functionalgastrointestinal disorder, gastroesophageal reflux disease, functionalheartburn, dyspepsia, functional dyspepsia, nonulcer dyspepsia,gastroparesis, chronic intestinal pseudo-obstruction, colonicpseudo-obstruction), obesity, congestive heart failure or benignprostatic hyperplasia by administering a composition comprising anaforementioned peptide

When Xaa₉ is Trp, Tyr or Phe or when Xaa₁₆ is Trp the peptide has apotentially functional chymotrypsin cleavage site that is located at aposition where cleavage will inactivate GC-C receptor binding by thepeptide. When Xaa₉ is Lys or Arg or when Xaa₁₆ is Lys or Arg, thepeptide has a potentially functional trypsin cleavage site that islocated at a position where cleavage will inactivate GC-C receptorbinding by the peptide.

When Xaa₁₉ is Trp, Tyr or Phe, the peptide has a chymotrypsin cleavagesite that is located at a position where cleavage will liberate theportion of the peptide carboxy-terminal to Xaa₁₉. When Xaa₁₉ is Leu, Ileor Val, the peptide can have a chymotrypsin cleavage site that islocated at a position where cleavage will liberate the portion of thepeptide amino-terminal to Xaa₁₉. At relatively high pH the same effectis seen when Xaa₁₉ is His. When Xaa₁₉ is Lys or Arg, the peptide has atrypsin cleavage site that is located at a position where cleavage willliberate portion of the peptide carboxy-terminal to Xaa₁₉. Thus, if thepeptide includes an analgesic peptide carboxy-terminal to Xaa₁₉, thepeptide will be liberated in the digestive tract upon exposure to theappropriate protease. Among the analgesic peptides which can be includedin the peptide are: AspPhe (as Xaa₂₀ Xaa₂₁), endomorphin-1,endomorphin-2, nocistatin, dalargin, lupron, and substance P and otheranalgesic peptides described herein. These peptides can, for example, beused to replace Xaa₂₀ Xaa₂₁.

When Xaa₁ or the amino-terminal amino acid of the peptide of theinvention (e.g., Xaa₂ or Xaa₃) is Trp, Tyr or Phe, the peptide has achymotrypsin cleavage site that is located at a position where cleavagewill liberate the portion of the peptide amino-terminal to Xaa₁ (or Xaa₂or Xaa₃) along with Xaa₁, Xaa₂ or Xaa₃. When Xaa₁ or the amino-terminalamino acid of the peptide of the invention (e.g., Xaa₂ or Xaa₃) is Lysor Arg, the peptide has a trypsin cleavage site that is located at aposition where cleavage will liberate portion of the peptideamino-terminal to Xaa₁ along with Xaa₁, Xaa₂ or Xaa₃). When Xaa₁ or theamino-terminal amino acid of the peptide of the invention is Leu, Ile orVal, the peptide can have a chymotrypsin cleavage site that is locatedat a position where cleavage will liberate the portion of the peptideamino-terminal to Xaa₁. At relatively high pH the same effect is seenwhen Xaa₁ is His. Thus, for example, if the peptide includes ananalgesic peptide amino-terminal to Xaa₁, the peptide will be liberatedin the digestive tract upon exposure to the appropriate protease. Amongthe analgesic peptides which can be included in the peptide are: AspPhe,endomorphin-1, endomorphin-2, nocistatin, dalargin, lupron, andsubstance p and other analgesic peptides described herein.

When fully folded, disulfide bonds are present between: Cys₆ and Cys₁₁;Cys₇ and Cys₁₅; and Cys₁₀ and Cys₁₈. The peptides of the invention bearsome sequence similarity to ST peptides. However, they include aminoacid changes and/or additions that improve functionality. These changescan, for example, increase or decrease activity (e.g., increase ordecrease the ability of the peptide to stimulate intestinal motility),alter the ability of the peptide to fold correctly, the stability of thepeptide, the ability of the peptide to bind the GC-C receptor and/ordecrease toxicity. In some cases the peptides may function moredesirably than wild-type ST peptide. For example, they may limitundesirable side effects such as diarrhea and dehydration.

In some embodiments one or both members of one or more pairs of Cysresidues which normally form a disulfide bond can be replaced byhomocysteine, 3-mercaptoproline (Kolodziej et al. 1996 Int J PeptProtein Res 48:274); β, β dimethylcysteine (Hunt et al. 1993 Int J PeptProtein Res 42:249) or diaminopropionic acid (Smith et al. 1978 J MedChem 21:117) to form alternative internal cross-links at the positionsof the normal disulfide bonds.

In addition, one or more disulfide bonds can be replaced by alternativecovalent cross-links, e.g., an amide bond, an ester linkage, an alkyllinkage, a thio ester linkage, a lactam bridge, a carbamoyl linkage, aurea linkage, a thiourea linkage, a phosphonate ester linkage, an alkyllinkage, and alkenyl linkage, an ether, a thioether linkage, or an aminolinkage. For example, Ledu et al. (Proceedings Nat'l Acad. Sci.100:11263-78, 2003) described methods for preparing lactam and amidecross-links. Schafmeister et al. (J. Am. Chem. Soc. 122:5891, 2000)describes stable, all carbon cross-links. In some cases, the generationof such alternative cross-links requires replacing the Cys residues withother residues such as Lys or Glu or non-naturally occurring aminoacids.

In the case of a peptide comprising the sequence (I): Xaa₁ Xaa₂ Xaa₃Xaa₄ Xaa₅ Cys₆ Cys₇ Xaa₈ Xaa₉ Cys₁₀ Cys₁₁ Xaa₁₂ Xaa₁₃ Xaa₁₄ Cys₁₅ Xaa₁₆Xaa₁₇ Cys₁₈ Xaa₁₉ Xaa₂₀ Xaa₂₁ wherein: Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ ismissing and/or the sequence Xaa₁₉ Xaa₂₀ Xaa₂₁ is missing, the peptidecan still contain additional carboxyterminal or amino terminal aminoacids or both. For example, the peptide can include an amino terminalsequence that facilitates recombinant production of the peptide and iscleaved prior to administration of the peptide to a patient. The peptidecan also include other amino terminal or carboxyterminal amino acids. Insome cases the additional amino acids protect the peptide, stabilize thepeptide or alter the activity of the peptide. In some cases some or allof these additional amino acids are removed prior to administration ofthe peptide to a patient. The peptide can include 1, 2, 3, 4, 5, 10, 15,20, 25, 30, 40, 50, 60, 70 80, 90, 100 or more amino acids at its aminoterminus or carboxy terminus or both. The number of flanking amino acidsneed not be the same. For example, there can be 10 additional aminoacids at the amino terminus of the peptide and none at the carboxyterminus.

In one embodiment the peptide comprises the amino acid sequence (I):Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Cys₆ Cys₇ Xaa₈ Xaa₉ Cys₁₀ Cys₁₁ Xaa₁₂ Xaa₁₃Xaa₁₄ Cys₁₅ Xaa₁₆ Xaa₁₇ Cys₁₈ Xaa₁₉ Xaa₂₀ Xaa₂₁ (SEQ ID NO:144) wherein:Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ is missing; Xaa₈ is Glu; Xaa₉ is Leu, Ile, Lys,Arg, Trp, Tyr or Phe; Xaa₁₂ is Asn; Xaa₁₃ is Pro; Xaa₁₄ is Ala; Xaa₁₆ isThr, Ala, Lys, Arg, Trp; Xaa₁₇ is Gly; Xaa₁₉ is Tyr or Leu; and Xaa₂₀Xaa₂₁ is AspPhe or is missing. Where Xaa₂₀ Xaa₂₁ and/or Xaa₁ Xaa₂ Xaa₃Xaa₄ Xaa₅ are missing, there may be additional flanking amino acids insome embodiments. In certain embodiments, the peptide does not consistof any of the peptides of Table I

In a second aspect, the invention also features a therapeutic orprophylactic method comprising administering a peptide comprising theamino acid sequence (I): Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Cys₆ Cys₇ Xaa₈ Xaa₉Cys₁₀ Cys₁₁ Xaa₁₂ Xaa₁₃ Xaa₁₄ Cys₁₅ Xaa₁₆ Xaa₁₇ Cys₁₈ Xaa₁₉ Xaa₂₀ Xaa₂₁(SEQ ID NO:145) wherein: Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ is Asn Ser Ser Asn Tyr(SEQ ID NO:121) or is missing or Xaa₁ Xaa₂ Xaa₃ Xaa₄ is missing and Xaa₅is Asn, Trp, Tyr, Asp, Ile, Thr, or Phe; Xaa₈ is Glu, Asp, Gln, Gly orPro; Xaa₉ is Leu, Ile, Val, Ala, Lys, Arg, Trp, Tyr or Phe; Xaa₁₂ isAsn, Tyr, Asp or Ala; Xaa₁₃ is Pro or Gly; Xaa₁₄ is Ala, Leu, Ser, Gly,Val, Glu, Gln, Ile, Leu, Lys, Arg, and Asp; Xaa₁₆ is Thr, Ala, Asn, Lys,Arg, Trp; Xaa₁₇ is Gly, Pro or Ala; Xaa₁₉ is Trp, Tyr, Phe or Leu; andXaa₂₀ Xaa₂₁ is AspPhe or is missing or Xaa₂₀ is Asn or Glu and Xaa₂₁ ismissing or Xaa₁₉ Xaa₂₀ Xaa₂₁ is missing.

In certain embodiments of the therapeutic or prophylactic methods: thepeptide comprises the amino acid sequence (I): Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅Cys₆ Cys₇ Xaa₈ Xaa₉ Cys₁₀ Cys₁₁ Xaa₁₂ Xaa₁₃ Xaa₁₄ Cys₁₅ Xaa₁₆ Xaa₁₇Cys₁₈ Xaa₁₉ Xaa₂₀ Xaa₂₁ (SEQ ID NO:146) wherein: Xaa₁ Xaa₂ Xaa₃ Xaa₄Xaa₅ is missing; Xaa₈ is Glu; Xaa₉ is Leu, Ile, Lys, Arg, Trp, Tyr, orPhe; Xaa₁₂ is Asn; Xaa₁₃ is Pro; Xaa₁₄ is Ala; Xaa₁₆ is Thr, Ala, Lys,Arg, Trp or Xaa₁₆ is any amino acid or Xaa₁₆ is Thr, Ala, Lys, Arg, Trpor Xaa₁₆ is any non-aromatic amino acid; Xaa₁₇ is Gly; Xaa₁₉ is Tyr orLeu; and Xaa₂₀ Xaa₂₁ is AspPhe or is missing.

In certain embodiments, the invention features, a purified polypeptidecomprising the amino acid sequence (II):

Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Cys₆ Cys₇ Xaa₈ Xaa₉ Cys₁₀ Cys₁₁ Asn₁₂ Pro₁₃Ala₁₄ Cys₁₅ Xaa₁₆ Gly₁₇ Cys₁₈ Xaa₁₉ Xaa₂₀ Xaa₂₁ (SEQ ID NO:120) wherein

Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ is Asn Ser Ser Asn Tyr (SEQ ID NO:121) or ismissing or Xaa₁ Xaa₂ Xaa₃ Xaa₄ is missing and Xaa₅ is Asn;

Xaa₈ is Glu or Asp;

Xaa₉ is Leu, Ile, Val, Trp, Tyr or Phe;

Xaa₁₆ is Thr, Ala, Trp;

Xaa₁₉ is Trp, Tyr, Phe or Leu or is missing; and Xaa₂₀ Xaa₂₁ is AspPhe.

In various preferred embodiments the invention features a purifiedpolypeptide comprising the amino acid sequence (II): Xaa₁ Xaa₂ Xaa₃ Xaa₄Xaa₅ Cys₆ Cys₇ Xaa₈ Xaa₉ Cys₁₀ Cys₁₁ Asn₁₂ Pro₁₃ Ala₁₄Cys₁₅ Xaa₁₆ Gly₁₇Cys₁₈ Xaa₁₉ Xaa₂₀ Xaa₂₁ (SEQ ID NO:149) wherein, Xaa₉ is Leu, Ile or Valand Xaa₁₆ is Trp, Tyr or Phe; Xaa₉ is Trp, Tyr or Phe, and Xaa₁₆ is Thror Ala; Xaa₁₉ is Trp, Tyr, Phe and Xaa₂₀ Xaa₂₁ is AspPhe; and Xaa₁ Xaa₂Xaa₃ Xaa₄ is missing and Xaa₅ is Asn; the peptide comprises fewer than50, 40, 30 or 25 amino acids; fewer than five amino acid precede Cys₆.

The peptides can be co-administered with or linked, e.g., covalentlylinked to any of a variety of other peptides including analgesicpeptides or analgesic compounds. For example, a therapeutic peptide ofthe invention can be linked to an analgesic agent selected from thegroup consisting of: Ca channel blockers (e.g., ziconotide), complete orpartial 5HT receptor antagonists (for example 5HT3 (e.g. alosetron,ATI-7000; Aryx Thearpeutics, Santa Clara Calif.), 5HT4 and 5HT1 receptorantagonists), complete or partial 5HT receptor agonists including 5HT3,5HT4 (e.g. tegaserod, mosapride and renzapride) and 5HT1 receptoragonists, CRF receptor agonists (NBI-34041), β-3 adrenoreceptoragonists, opioid receptor agonists (e.g., loperamide, fedotozine, andfentanyl, naloxone, naltrexone, methyl nalozone, nalmefene, cypridime,beta funaltrexamine, naloxonazine, naltrindole, and nor-binaltorphimine,morphine, diphenyloxylate, enkephalin pentapeptide, asimadoline, andtrimebutine), NK1 receptor antagonists (e.g., ezlopitant and SR-14033),CCK receptor agonists (e.g., loxiglumide), NK1 receptor antagonists, NK3receptor antagonists (e.g., talnetant, osanetant (SR-142801),SSR-241586), norepinephrine-serotonin reuptake inhibitors (NSRI; e.g.,milnacipran), opiod receptor antagonists (e.g. naltrexone) vanilloid andcannabanoid receptor agonists (e.g., arvanil), sialorphin,sialorphin-related peptides comprising the amino acid sequence QHNPR(SEQ ID NO:111) for example, VQHNPR (SEQ ID NO:112); VRQHNPR (SEQ IDNO:113); VRGQHNPR (SEQ ID NO:114); VRGPQHNPR (SEQ ID NO:115); VRGPRQHNPR(SEQ ID NO:116); VRGPRRQHNPR (SEQ ID NO:117); and RQHNPR (SEQ IDNO:118), compounds or peptides that are inhibitors of neprilysin,frakefamide (H-Tyr-D-Ala-Phe(F)-Phe-NH₂; WO 01/019849 A1), loperamide,Tyr-Arg (kyotorphin), CCK receptor agonists (caerulein), conotoxinpeptides, peptide analogs of thymulin, loxiglumide, dexloxiglumide (theR-isomer of loxiglumide) (WO 88/05774) and other analgesic peptides orcompounds can be used with or linked to the peptides of the invention.

Amino acid, non-amino acid, peptide and non-peptide spacers can beinterposed between a peptide that is a GC-C receptor agonist and apeptide that has some other biological function, e.g., an analgesicpeptide or a peptide used to treat obesity. The linker can be one thatis cleaved from the flanking peptides in vivo or one that remains linkedto the flanking peptides in vivo. For example, glycine, beta-alanine,glycyl-glycine, glycyl-beta-alanine, gamma-aminobutyric acid,6-aminocaproic acid, L-phenylalanine, L-tryptophan andglycil-L-valil-L-phenylalanine can be used as a spacer (Chaltin et al.2003 Helvetica Chimica Acta 86:533-547; Caliceti et al. 1993 FARMCO48:919-32) as can polyethylene glycols (Butterworth et al. 1987 J. Med.Chem 30:1295-302) and maleimide derivatives (King et al. 2002Tetrahedron Lett. 43:1987-1990). Various other linkers are described inthe literature (Nestler 1996 Molecular Diversity 2:35-42; Finn et al.1984 Biochemistry 23:2554-8; Cook et al. 1994 Tetrahedron Lett.35:6777-80; Brokx et al. 2002 Journal of Controlled Release 78:115-123;Griffin et al. 2003 J. Am. Chem. Soc. 125:6517-6531; Robinson et al.1998 Proc. Natl. Acad. Sci. USA 95:5929-5934.

The peptides can include the amino acid sequence of a peptide thatoccurs naturally in a vertebrate (e.g., mammalian) species or in abacterial species. In addition, the peptides can be partially orcompletely non-naturally occurring peptides. Also within the inventionare peptidomimetics corresponding to the peptides of the invention. Invarious embodiments, the patient is suffering from a gastrointestinaldisorder; the patient is suffering from a disorder selected from thegroup consisting of: a gastrointestinal motility disorder, irritablebowel syndrome, chronic constipation, a functional gastrointestinaldisorder, gastroesophageal reflux disease, functional heartburn,dyspepsia, functional dyspepsia, nonulcer dyspepsia, gastroparesis,chronic intestinal pseudo-obstruction, Crohn's disease, ulcerativecolitis, Irritable bowel syndrome, colonic pseudo-obstruction, obesity,congestive heart failure, or benign prostatic hyperplasia; thecomposition is administered orally; the peptide comprises 30 or feweramino acids, the peptide comprises 20 or fewer amino acids, and thepeptide comprises no more than 5 amino acids prior to Cys₆; the peptidecomprises 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, or 30 orfewer amino acids. In other embodiments, the peptide comprises 20 orfewer amino acids. In other embodiments the peptide comprises no morethan 20, 15, 10, or 5 peptides subsequent to Cys₁₈. In certainembodiments Xaa₁₉ is a chymotrypsin or trypsin cleavage site and ananalgesic peptide is present immediately following Xaa₁₉.

In a third aspect, the invention features a method for treating apatient suffering from constipation. Clinically accepted criteria thatdefine constipation range from the frequency of bowel movements, theconsistency of feces and the ease of bowel movement. One commondefinition of constipation is less than three bowel movements per week.Other definitions include abnormally hard stools or defecation thatrequires excessive straining (Schiller 2001, Aliment Pharmacol Ther15:749-763). Constipation may be idiopathic (functional constipation orslow transit constipation) or secondary to other causes includingneurologic, metabolic or endocrine disorders. These disorders includediabetes mellitus, hypothyroidism, hyperthyroidism, hypocalcaemia,Multiple Sclerosis, Parkinson's disease, spinal cord lesions,Neurofibromatosis, autonomic neuropathy, Chagas disease, Hirschsprung'sdisease and Cystic fibrosis. Constipation may also be the result ofsurgery (postoperative ileus) or due to the use of drugs such asanalgesics (like opiods), antihypertensives, anticonvulsants,antidepressants, antispasmodics and antipsychotics.

The method comprising administering a composition comprising a purifiedpolypeptide comprising the amino acid sequence (I): Xaa₁ Xaa₂ Xaa₃ Xaa₄Xaa₅ Cys₆ Cys₇ Xaa₈ Xaa₉ Cys₁₀ Cys₁₁ Xaa₁₂ Xaa₁₃ Xaa₁₄ Cys₁₅ Xaa₁₆ Xaa₁₇Cys₁₈ Xaa₁₉ Xaa₂₀ Xaa₂₁ (SEQ ID NO:147) wherein: Xaa₁ Xaa₂ Xaa₃ Xaa₄Xaa₅ is Asn Ser Ser Asn Tyr (SEQ ID NO:121) or is missing or Xaa₁ Xaa₂Xaa₃ Xaa₄ is missing and Xaa₅ is Asn, Trp, Tyr, Asp, Ile, Thr, or Phe;Xaa₈ is Glu, Asp, Gln, Gly or Pro; Xaa₉ is Leu, Ile, Val, Ala, Lys, Arg,Trp, Tyr or Phe; Xaa₁₂ is Asn, Tyr, Asp or Ala; Xaa₁₃ is Pro or Gly;Xaa₁₄ is Ala, Leu, Ser, Gly, Val, Glu, Gln, Ile, Leu, Lys, Arg, and Asp;Xaa₁₆ is Thr, Ala, Asn, Lys, Arg, Trp; Xaa₁₇ is Gly, Pro or Ala; Xaa₁₉is Trp, Tyr, Phe or Leu; Xaa₁₉ is Lys or Arg; Xaa₂₀ Xaa₂₁ is AspPhe oris missing or Xaa₂₀ is Asn or Glu and Xaa₂₁ is missing or Xaa₁₉ Xaa₂₀Xaa₂₁ is missing.

In one embodiment of the method, the peptide comprises the amino acidsequence (I): Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Cys₆ Cys₇ Xaa₈ Xaa₉ Cys₁₀ Cys₁₁Xaa₁₂ Xaa₁₃ Xaa₁₄ Cys₁₅ Xaa₁₆ Xaa₁₇ Cys₁₈ Xaa₁₉ Xaa₂₀ Xaa₂₁ (SEQ IDNO:148) wherein: Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ is missing; Xaa₈ is Glu; Xaa₉is Leu, Ile, Lys, Arg, Trp, Tyr or Phe; Xaa₁₂ is Asn; Xaa₁₃ is Pro;Xaa₁₄ is Ala; Xaa₁₆ is Thr, Ala, Lys, Arg, Trp; Xaa₁₇ is Gly; Xaa₁₉ isTyr or Leu; Xaa₁₉ is Lys or Arg; Xaa₂₀ Xaa₂₁ is AspPhe or is missing.

In various preferred embodiments, the constipation is associated withuse of a therapeutic agent; the constipation is associated with aneuropathic disorder; the constipation is post-surgical constipation(postoperative ileus); and the constipation associated with agastrointestinal disorder; the constipation is idiopathic (functionalconstipation or slow transit constipation); the constipation isassociated with neuropathic, metabolic or endocrine disorder (e.g.,diabetes mellitus, hypothyroidism, hyperthyroidism, hypocalcaemia,Multiple Sclerosis, Parkinson's disease, spinal cord lesions,neurofibromatosis, autonomic neuropathy, Chagas disease, Hirschsprung'sdisease or cystic fibrosis). Constipation may also be the result ofsurgery (postoperative ileus) or due the use of drugs such as analgesics(e.g., opiods), antihypertensives, anticonvulsants, antidepressants,antispasmodics and antipsychotics.

In a fourth aspect, the invention features a method for treating apatient suffering a gastrointestinal disorder, the method comprisingadministering to the patient a composition comprising a purifiedpolypeptide comprising the amino acid sequence (I): Xaa₁ Xaa₂ Xaa₃ Xaa₄Xaa₅ Cys₆ Cys₇ Xaa₈ Xaa₉ Cys₁₀ Cys₁₁ Xaa₁₂ Xaa₁₃ Xaa₁₄ Cys₁₅ Xaa₁₆ Xaa₁₇Cys₁₈ Xaa₁₉ Xaa₂₀ Xaa₂₁ (SEQ ID NO:147) wherein: Xaa₁ Xaa₂ Xaa₃ Xaa₄Xaa₅ is Asn Ser Ser Asn Tyr (SEQ ID NO:121) or is missing or Xaa₁ Xaa₂Xaa₃ Xaa₄ is missing and Xaa₅ is Asn, Trp, Tyr, Asp, Ile, Thr, or Phe;Xaa₈ is Glu, Asp, Gln, Gly or Pro; Xaa₉ is Leu, Ile, Val, Ala, Lys, Arg,Trp, Tyr or Phe; Xaa₁₂ is Asn, Tyr, Asp or Ala; Xaa₁₃ is Pro or Gly;Xaa₁₄ is Ala, Leu, Ser, Gly, Val, Glu, Gln, Ile, Leu, Lys, Arg, and Asp;Xaa₁₆ is Thr, Ala, Asn, Lys, Arg, Trp; Xaa₁₇ is Gly, Pro or Ala; Xaa₁₉is Trp, Tyr, Phe or Leu; Xaa₁₉ is Lys or Arg; Xaa₂₀ Xaa₂₁ is AspPhe oris missing or Xaa₂₀ is Asn or Glu and Xaa₂₁ is missing or Xaa₁₉ Xaa₂₀Xaa₂₁ is missing.

In one embodiment of the method, the peptide comprises the amino acidsequence (I): Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Cys₆ Cys₇ Xaa₈ Xaa₉ Cys₁₀ Cys₁₁Xaa₁₂ Xaa₁₃ Xaa₁₄ Cys₁₅ Xaa₁₆ Xaa₁₇ Cys₁₈ Xaa₁₉ Xaa₂₀ Xaa₂₁ (SEQ ID NO:148) wherein: Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ is missing; Xaa₈ is Glu; Xaa₉ isLeu, Ile, Lys, Arg, Trp, Tyr or Phe; Xaa₁₂ is Asn; Xaa₁₃ is Pro; Xaa₁₄is Ala; Xaa₁₆ is Thr, Ala, Lys, Arg, Trp; Xaa₁₇ is Gly; Xaa₁₉ is Tyr orLeu; Xaa₁₉ is Lys or Arg; Xaa₂₀ Xaa₂₁ is AspPhe or is missing.

In various embodiments, the patient is suffering from a gastrointestinaldisorder; the patient is suffering from a disorder selected from thegroup consisting of: a gastrointestinal motility disorder, irritablebowel syndrome, chronic constipation, a functional gastrointestinaldisorder, gastroesophageal reflux disease, functional heartburn,dyspepsia, functional dyspepsia, nonulcer dyspepsia, gastroparesis,chronic intestinal pseudo-obstruction, Crohn's disease, ulcerativecolitis, Inflammatory bowel disease, colonic pseudo-obstruction,obesity, congestive heart failure, or benign prostatic hyperplasia.

In various preferred embodiments, Xaa₉ is Leu, Ile or Val and Xaa₁₆ isTrp, Tyr or Phe; Xaa₉ is Trp, Tyr or Phe and Xaa₁₆ is Thr or Ala; Xaa₁₉is Trp, Tyr, Phe; Xaa₁₉ is Lys or Arg; Xaa₂₀ Xaa₂₁ is AspPhe; Xaa₁ Xaa₂Xaa₃ Xaa₄ is missing and Xaa₅ is Asn.

In a fifth aspect, the invention features a method for increasinggastrointestinal motility in a patient, the method comprising:administering to the patient a composition comprising a purifiedpolypeptide comprising the amino acid sequence (I): Xaa₁ Xaa₂ Xaa₃ Xaa₄Xaa₅ Cys₆ Cys₇ Xaa₈ Xaa₉ Cys₁₀ Cys₁₁ Xaa₁₂ Xaa₁₃ Xaa₁₄ Cys₁₅ Xaa₁₆ Xaa₁₇Cys₁₈ Xaa₁₉ Xaa₂₀ Xaa₂₁ (SEQ ID NO:147) wherein: Xaa₁ Xaa₂ Xaa₃ Xaa₄Xaa₅ is Asn Ser Ser Asn Tyr (SEQ ID NO:121) or is missing or Xaa₁ Xaa₂Xaa₃ Xaa₄ is missing and Xaa₅ is Asn, Trp, Tyr, Asp, Ile, Thr, or Phe;Xaa₈ is Glu, Asp, Gln, Gly or Pro; Xaa₉ is Leu, Ile, Val, Ala, Lys, Arg,Trp, Tyr or Phe; Xaa₁₂ is Asn, Tyr, Asp or Ala; Xaa₁₃ is Pro or Gly;Xaa₁₄ is Ala, Leu, Ser, Gly, Val, Glu, Gln, Ile, Leu, Lys, Arg, and Asp;Xaa₁₆ is Thr, Ala, Asn, Lys, Arg, Trp; Xaa₁₇ is Gly, Pro or Ala; Xaa₁₉is Trp, Tyr, Phe or Leu; Xaa₁₉ is Lys or Arg; Xaa₂₀ Xaa₂₁ is AspPhe oris missing or Xaa₂₀ is Asn or Glu and Xaa₂₁ is missing or Xaa₁₉ Xaa₂₀Xaa₂₁ is missing.

In one embodiment the peptide comprises the amino acid sequence (I):Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Cys₆ Cys₇ Xaa₈ Xaa₉ Cys₁₀ Cys₁₁ Xaa₁₂ Xaa₁₃Xaa₁₄ Cys₁₅ Xaa₁₆ Xaa_(r) Cys₁₈ Xaa₁₉ Xaa₂₀ Xaa₂₁ (SEQ ID NO:148)wherein: Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ is missing; Xaa₈ is Glu; Xaa₉ is Leu,Ile, Lys, Arg, Trp, Tyr or Phe; Xaa₁₂ is Asn; Xaa₁₃ is Pro; Xaa₁₄ isAla; Xaa₁₆ is Thr, Ala, Lys, Arg, Trp; Xaa₁₇ is Gly; Xaa₁₉ is Tyr orLeu; Xaa₁₉ is Lys or Arg; Xaa₂₀ Xaa₂₁ is AspPhe or is missing.

In a sixth aspect, the invention features a method for increasing theactivity of an intestinal guanylate cyclase (GC-C) receptor in apatient, the method comprising: administering to the patient acomposition comprising a purified polypeptide comprising the amino acidsequence (I): Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Cys₆ Cys₇ Xaa₈ Xaa₉ Cys₁₀ Cys₁₁Xaa₁₂ Xaa₁₃ Xaa₁₄ Cys₁₅ Xaa₁₆ Xaa₁₇ Cys₁₈ Xaa₁₉ Xaa₂₀ Xaa₂₁ (SEQ IDNO:147) wherein: Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ is Asn Ser Ser Asn Tyr (SEQ IDNO:121) or is missing or Xaa₁ Xaa₂ Xaa₃ Xaa₄ is missing and Xaa₅ is Asn,Trp, Tyr, Asp, Ile, Thr, or Phe; Xaa₈ is Glu, Asp, Gln, Gly or Pro; Xaa₉is Leu, Ile, Val, Ala, Lys, Arg, Trp, Tyr or Phe; Xaa₁₂ is Asn, Tyr, Aspor Ala; Xaa₁₃ is Pro or Gly; Xaa₁₄ is Ala, Leu, Ser, Gly, Val, Glu, Gln,Ile, Leu, Lys, Arg, and Asp; Xaa₁₆ is Thr, Ala, Asn, Lys, Arg, Trp;Xaa₁₇ is Gly, Pro or Ala; Xaa₁₉ is Trp, Tyr, Phe or Leu; Xaa₁₉ is Lys orArg; Xaa₂₀ Xaa₂₁ is AspPhe or is missing or Xaa₂₀ is Asn or Glu andXaa₂₁ is missing or Xaa₁₉ Xaa₂₀ Xaa₂₁ is missing.

In one embodiment the peptide comprises the amino acid sequence (I):Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Cys₆ Cys₇ Xaa₈ Xaa₉ Cys₁₀ Cys₁₁ Xaa₁₂ Xaa₁₃Xaa₁₄ Cys₁₅ Xaa₁₆ Xaa₁₇ Cys₁₈ Xaa₁₉ Xaa₂₀ Xaa₂₁ (SEQ ID NO:148) wherein:Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ is missing; Xaa₈ is Glu; Xaa₉ is Leu, Ile, Lys,Arg, Trp, Tyr or Phe; Xaa₁₂ is Asn; Xaa₁₃ is Pro; Xaa₁₄ is Ala; Xaa₁₆ isThr, Ala, Lys, Arg, Trp; Xaa₁₇ is Gly; Xaa₁₉ is Tyr or Leu; Xaa₁₉ is Lysor Arg; Xaa₂₀ Xaa₂₁ is AspPhe or is missing.

In a seventh aspect, the invention features an isolated nucleic acidmolecule comprising a nucleotide sequence encoding a polypeptidecomprising the amino acid sequence: (I): Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Cys₆Cys₇ Xaa₈ Xaa₉ Cys₁₀ Cys₁₁ Xaa₁₂ Xaa₁₃ Xaa₁₄ Cys₁₅ Xaa₁₆ Xaa₁₇ Cys₁₈Xaa₁₉ Xaa₂₀ Xaa₂₁ (SEQ ID NO:147) wherein: Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ isAsn Ser Ser Asn Tyr (SEQ ID NO:121) or is missing or Xaa₁ Xaa₂ Xaa₃ Xaa₄is missing and Xaa₅ is Asn, Trp, Tyr, Asp, Ile, Thr, or Phe; Xaa₈ isGlu, Asp, Gln, Gly or Pro; Xaa₉ is Leu, Ile, Val, Ala, Lys, Arg, Trp,Tyr or Phe; Xaa₁₂ is Asn, Tyr, Asp or Ala; Xaa₁₃ is Pro or Gly; Xaa₁₄ isAla, Leu, Ser, Gly, Val, Glu, Gln, Ile, Leu, Lys, Arg, and Asp; Xaa₁₆ isThr, Ala, Asn, Lys, Arg, Trp; Xaa₁₇ is Gly, Pro or Ala; Xaa₁₉ is Trp,Tyr, Phe or Leu; Xaa₁₉ is Lys or Arg; Xaa₂₀ Xaa₂₁ is AspPhe or ismissing or Xaa₂₀ is Asn or Glu and Xaa₂₁ is missing or Xaa₁₉ Xaa₂₀ Xaa₂₁is missing.

In one embodiment the peptide comprises the amino acid sequence (I):Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Cys₆ Cys₇ Xaa₈ Xaa₉ Cys₁₀ Cys₁₁ Xaa₁₂ Xaa₁₃Xaa₁₄ Cys₁₅ Xaa₁₆ Xaa₁₇ Cys₁₈) Xaa₁₉ Xaa₂₀ Xaa₂₁ (SEQ ID NO:148)wherein: Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ is missing; Xaa₈ is Glu; Xaa₉ is Leu,Ile, Lys, Arg, Trp, Tyr or Phe; Xaa₁₂ is Asn; Xaa₁₃ is Pro; Xaa₁₄ isAla; Xaa₁₆ is Thr, Ala, Lys, Arg, Trp; Xaa₁₇ is Gly; Xaa₁₉ is Tyr orLeu; Xaa₁₉ is Lys or Arg; Xaa₂₀ Xaa₂₁ is AspPhe or is missing.

In an eighth aspect the invention features a method for treatingconstipation, the method comprising administering an agonist of theintestinal guanylate cyclase (GC-C) receptor. In various embodiments:the agonist is a peptide, the peptide includes four Cys that form twodisulfide bonds, and the peptide includes six Cys that form threedisulfide bonds.

In a ninth aspect, the invention features a method for treating agastrointestinal disorder, a gastrointestinal motility disorder,irritable bowel syndrome, chronic constipation, a functionalgastrointestinal disorder, gastroesophageal reflux disease, functionalheartburn, dyspepsia, functional dyspepsia, nonulcer dyspepsia,gastroparesis, chronic intestinal pseudo-obstruction, colonicpseudo-obstruction, Crohn's disease, ulcerative colitis, Inflammatorybowel disease, obesity, congestive heart failure, or benign prostatichyperplasia, the method comprising administering an agonist of theintestinal guanylate cyclase (GC-C) receptor either orally, by rectalsuppository, or parenterally. In various embodiments: the agonist is apeptide, the peptide includes four Cys that form two disulfide bonds,and the peptide includes six Cys that form three disulfide bonds.

In a tenth aspect, the invention features a method for treating agastrointestinal disorder selected from the group consisting of: agastrointestinal motility disorder, irritable bowel syndrome, chronicconstipation, a functional gastrointestinal disorder, gastroesophagealreflux disease, functional heartburn, dyspepsia, functional dyspepsia,nonulcer dyspepsia, gastroparesis, chronic intestinalpseudo-obstruction, colonic pseudo-obstruction, Crohn's disease,ulcerative colitis, Inflammatory bowel disease, the method comprisingadministering an agonist of the intestinal guanylate cyclase (GC-C)receptor. In various embodiments the composition is administered orally;the peptide comprises 30 or fewer amino acids, the peptide comprises 20or fewer amino acids, and the peptide comprises no more than 5 aminoacids prior to Cys₅.

In various embodiments: the agonist is a peptide, the peptide includesfour Cys that form two disulfide bonds, and the peptide includes six Cysthat form three disulfide bonds.

In an eleventh aspect, the invention features a method for treatingobesity, the method comprising administering an agonist of theintestinal guanylate cyclase (GC-C) receptor. In various embodiments:the agonist is a peptide, the peptide includes four Cys that form twodisulfide bonds, and the peptide includes six Cys that form threedisulfide bonds.

In a twelfth aspect, the invention features a method for treatingobesity, the method comprising administering a polypeptide comprisingthe amino acid sequence: (I): Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Cys₆ Cys₇ Xaa₈Xaa₉ Cys₁₀ Cys₁₁ Xaa₁₂ Xaa₁₃ Xaa₁₄ Cys₁₅ Xaa₁₆ Xaa₁₇ Cys₁₈ Xaa₁₉ Xaa₂₀Xaa₂₁ (SEQ ID NO:147) wherein: Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ is Asn Ser SerAsn Tyr (SEQ ID NO:121) or is missing or Xaa₁ Xaa₂ Xaa₃ Xaa₄ is missingand Xaa₅ is Asn, Trp, Tyr, Asp, Ile, Thr, or Phe; Xaa₈ is Glu, Asp, Gln,Gly or Pro; Xaa₉ is Leu, Ile, Val, Ala, Lys, Arg, Trp, Tyr or Phe; Xaa₁₂is Asn, Tyr, Asp or Ala; Xaa₁₃ is Pro or Gly; Xaa₁₄ is Ala, Leu, Ser,Gly, Val, Glu, Gln, Ile, Leu, Lys, Arg, and Asp; Xaa₁₆ is Thr, Ala, Asn,Lys, Arg, Trp; Xaa₁₇ is Gly, Pro or Ala; Xaa₁₉ is Trp, Tyr, Phe or Leu;and Xaa₂₀ Xaa₂₁ is AspPhe or is missing or Xaa₂₀ is Asn or Glu and Xaa₂₁is missing or Xaa₁₉ Xaa₂₀ Xaa₂₁ is missing. The peptide can beadministered alone or in combination with another agent for thetreatment of obesity, e.g., sibutramine or another agent, e.g., an agentdescribed herein.

In one embodiment the peptide comprises the amino acid sequence (I):Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Cys₆ Cys₇ Xaa₈ Xaa₉ Cys₁₀ Cys₁₁ Xaa₁₂ Xaa₁₃Xaa₁₄ Cys₁₅ Xaa₁₆ Xaa₁₇ Cys₁₈ Xaa₁₉ Xaa₂₀ Xaa₂₁ (SEQ ID NO:148) wherein:Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ is missing; Xaa₈ is Glu; Xaa₉ is Leu, Ile, Lys,Arg, Trp, Tyr or Phe; Xaa₁₂ is Asn; Xaa₁₃ is Pro; Xaa₁₄ is Ala; Xaa₁₆ isThr, Ala, Lys, Arg, Trp; Xaa₁₇ is Gly; Xaa₁₉ is Tyr or Leu; and Xaa₂₀Xaa₂₁ is AspPhe or is missing.

In a thirteenth aspect, the invention features a pharmaceuticalcomposition comprising a polypeptide described herein.

In a fourteenth aspect, the invention features a method for treatingcongestive heart failure, the method comprising: administering to thepatient a composition comprising a purified polypeptide comprising theamino acid sequence (I): Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Cys₆ Cys₇ Xaa₈ Xaa₉Cys₁₀ Cys₁₁ Xaa₁₂ Xaa₁₃ Xaa₁₄ Cys₁₅ Xaa₁₆ Xaa₁₇ Cys₁₈ Xaa₁₉ Xaa₂₀ Xaa₂₁(SEQ ID NO:147) wherein: Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ is Asn Ser Ser Asn Tyr(SEQ ID NO:121) or is missing or Xaa₁ Xaa₂ Xaa₃ Xaa₄ is missing and Xaa₅is Asn, Trp, Tyr, Asp, Ile, Thr, or Phe; Xaa₈ is Glu, Asp, Gln, Gly orPro; Xaa₉ is Leu, Ile, Val, Ala, Lys, Arg, Trp, Tyr or Phe; Xaa₁₂ isAsn, Tyr, Asp or Ala; Xaa₁₃ is Pro or Gly; Xaa₁₄ is Ala, Leu, Ser, Gly,Val, Glu, Gln, Ile, Leu, Lys, Arg, and Asp; Xaa₁₆ is Thr, Ala, Asn, Lys,Arg, Trp; Xaa₁₇ is Gly, Pro or Ala; Xaa₁₉ is Trp, Tyr, Phe or Leu; andXaa₂₀ Xaa₂₁ is AspPhe or is missing or Xaa₂₀ is Asn or Glu and Xaa₂₁ ismissing or Xaa₁₉ Xaa₂₀ Xaa₂₁ is missing. The peptide can be administeredin combination with another agent for treatment of congestive heartfailure, for example, a natriuretic peptide such as atrial natriureticpeptide, brain natriuretic peptide or C-type natriuretic peptide), adiuretic, or an inhibitor of angiotensin converting enzyme.

In one embodiment the peptide comprises the amino acid sequence (I):Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Cys₆ Cys₇ Xaa₈ Xaa₉ Cys₁₀ Cys₁₁ Xaa₁₂ Xaa₁₃Xaa₁₄ Cys₁₅ Xaa₁₆ Xaa₁₇ Cys₁₈) Xaa₁₉ Xaa₂₀ Xaa₂₁ (SEQ ID NO:148)wherein: Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ is missing; Xaa₈ is Glu; Xaa₉ is Leu,Ile, Lys, Arg, Trp, Tyr or Phe; Xaa₁₂ is Asn; Xaa₁₃ is Pro; Xaa₁₄ isAla; Xaa₁₆ is Thr, Ala, Lys, Arg, Trp; Xaa₁₇ is Gly; Xaa₁₉ is Tyr orLeu; Xaa₁₉ is Lys or Arg; Xaa₂₀ Xaa₂₁ is AspPhe or is missing.

In a fifteenth aspect, the invention features a method for treatingbenign prostatic hyperplasia, the method comprising: administering tothe patient a composition comprising a purified polypeptide comprisingthe amino acid sequence (I): Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Cys₆ Cys₇ Xaa₈Xaa₉ Cys₁₀ Cys₁₁ Xaa₁₂ Xaa₁₃ Xaa₁₄ Cys₁₅ Xaa₁₆ Xaa₁₇ Cys₁₈ Xaa₁₉ Xaa₂₀Xaa₂₁ (SEQ ID NO:147) wherein: Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ is Asn Ser SerAsn Tyr (SEQ ID NO:121) or is missing or Xaa₁ Xaa₂ Xaa₃ Xaa₄ is missingand Xaa₅ is Asn, Trp, Tyr, Asp, Ile, Thr, or Phe; Xaa₈ is Glu, Asp, Gln,Gly or Pro; Xaa₉ is Leu, Ile, Val, Ala, Lys, Arg, Trp, Tyr or Phe; Xaa₁₂is Asn, Tyr, Asp or Ala; Xaa₁₃ is Pro or Gly; Xaa₁₄ is Ala, Leu, Ser,Gly, Val, Glu, Gln, Ile, Leu, Lys, Arg, and Asp; Xaa₁₆ is Thr, Ala, Asn,Lys, Arg, Trp; Xaa₁₇ is Gly, Pro or Ala; Xaa₁₉ is Trp, Tyr, Phe or Leu;Xaa₁₉ is Lys or Arg; Xaa₂₀ Xaa₂₁ is AspPhe or is missing or Xaa₂₀ is Asnor Glu and Xaa₂₁ is missing or Xaa₁₉ Xaa₂₀ Xaa₂₁ is missing.

The peptide can be administered in combination with another agent fortreatment of BPH, for example, a 5-alpha reductase inhibitor (e.g.,finasteride) or an alpha adrenergic inhibitor (e.g., doxazosine).

In one embodiment the peptide comprises the amino acid sequence (I):Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Cys₆ Cys₇ Xaa₈ Xaa₉ Cys₁₀ Cys₁₁ Xaa₁₂ Xaa₁₃Xaa₁₄ Cys₁₅ Xaa₁₆ Xaa₁₇ Cys₁₈ Xaa₁₉ Xaa₂₀ Xaa₂₁ (SEQ ID NO:148) wherein:Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ is missing; Xaa₈ is Glu; Xaa₉ is Leu, Ile, Lys,Arg, Trp, Tyr or Phe; Xaa₁₂ is Asn; Xaa₁₃ is Pro; Xaa₁₄ is Ala; Xaa₁₆ isThr, Ala, Lys, Arg, Trp; Xaa₁₇ is Gly; Xaa₁₉ is Tyr or Leu; and Xaa₂₀Xaa₂₁ is AspPhe or is missing.

In a sixteenth aspect, the invention features a method for treating orreducing pain, including visceral pain, pain associated with agastrointestinal disorder or pain associated with some other disorder,the method comprising: administering to a patient a compositioncomprising a purified polypeptide comprising the amino acid sequence(I): Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Cys₆ Cys₇ Xaa₈ Xaa₉ Cys₁₀ Cys₁₁ Xaa₁₂Xaa₁₃ Xaa₁₄ Cys₁₅ Xaa₁₆ Xaa₁₇ Cys₁₈ Xaa₁₉ Xaa₂₀ Xaa₂₁, e.g., a purifiedpolypeptide comprising an amino acid sequence disclosed herein.

In a seventeenth aspect, the invention features a method for treatinginflammation, including inflammation of the gastrointestinal tract,e.g., inflammation associated with a gastrointestinal disorder orinfection or some other disorder, the method comprising: administeringto a patient a composition comprising a purified polypeptide comprisingthe amino acid sequence (I): Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Cys₆ Cys₇ Xaa₈Xaa₉ Cys₁₀ Cys₁₁ Xaa₁₂ Xaa₁₃ Xaa₁₄ Cys₁₅ Xaa₁₆ Xaa₁₇ Cys₁₈ Xaa₁₉ Xaa₂₀Xaa₂₁, e.g., a purified polypeptide comprising an amino acid sequencedisclosed herein.

In certain embodiments the peptide includes a peptide comprising orconsisting of the amino acid sequence Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Cys CysGlu Xaa₉ Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Xaa₂₀ Xaa₂₁ (II) (SEQID NO:66) wherein Xaa₉ is any amino acid, wherein Xaa₉ is any amino acidother than Leu, wherein Xaa₉ is selected from Phe, Trp and Tyr; whereinXaa₉ is selected from any other natural or non-natural aromatic aminoacid, wherein Xaa₉ is Tyr; wherein Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ is Asn SerSer Asn Tyr; wherein Xaa₁, Xaa₂, Xaa₃, Xaa₄, and Xaa₅ are missing;wherein Xaa₁, Xaa₂, Xaa₃ and Xaa₄ are missing; wherein Xaa₁, Xaa₂ andXaa₃ are missing; wherein Xaa₁ and Xaa₂ are missing; wherein Xaa₁ ismissing; wherein Xaa₂₀ Xaa₂₁ is AspPhe or is missing or Xaa₂₀ is Asn orGlu and Xaa₂₁ is missing or Xaa₁₉ Xaa₂₀ Xaa₂₁ is missing; wherein Xaa₁Xaa₂ Xaa₃ Xaa₄ Xaa₅ and Tyr Xaa₂₀ Xaa₂₁ are missing. In the case of apeptide comprising the sequence (I): Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ Cys₆ Cys₇Xaa₈ Xaa₉ Cys₁₀ Cys₁₁ Xaa₁₂ Xaa₁₃ Xaa₁₄ Cys₁₅ Xaa₁₆ Xaa₁₇ Cys₁₈ Xaa₁₉Xaa₂₀ Xaa₂₁ wherein: Xaa₁ Xaa₂ Xaa₃ Xaa₄ Xaa₅ is missing and/or thesequence Xaa₁₉ Xaa₂₀ Xaa₂₁ is missing peptide can still containadditional carboxyterminal or amino terminal amino acids or both

Among the useful peptides are peptides comprising, consisting of orconsisting essentially of the amino acid sequence Xaa₁ Xaa₂ Xaa₃ Xaa₄Xaa₅ Cys Cys Glu Xaa₉ Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr Xaa₂₀Xaa₂₁ (SEQ ID NO:66) are the following peptides:

(SEQ ID NO: 67) Gln Ser Ser Asn Tyr Cys Cys Glu Tyr Cys  Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 68)Asn Thr Ser Asn Tyr Cys Cys Glu Tyr Cys  Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 69)Asn Leu Ser Asn Tyr Cys Cys Glu Tyr Cys  Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 70)Asn Ile Ser Asn Tyr Cys Cys Glu Tyr Cys  Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 71)Asn Ser Ser Gln Tyr Cys Cys Glu Tyr Cys  Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 72)Ser Ser Asn Tyr Cys Cys Glu Tyr Cys Cys  Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 73)Gln Ser Ser Gln Tyr Cys Cys Glu Tyr Cys  Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 74)Ser Ser Gln Tyr Cys Cys Glu Tyr Cys Cys  Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 75)Asn Ser Ser Asn Tyr Cys Cys Glu Ala Cys  Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 76)Asn Ser Ser Asn Tyr Cys Cys Glu Arg Cys  Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 77)Asn Ser Ser Asn Tyr Cys Cys Glu Asn Cys  Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 78)Asn Ser Ser Asn Tyr Cys Cys Glu Asp Cys  Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 79)Asn Ser Ser Asn Tyr Cys Cys Glu Cys Cys  Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 80)Asn Ser Ser Asn Tyr Cys Cys Glu Gln Cys  Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 81)Asn Ser Ser Asn Tyr Cys Cys Glu Glu Cys  Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 82)Asn Ser Ser Asn Tyr Cys Cys Glu Gly Cys  Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 83)Asn Ser Ser Asn Tyr Cys Cys Glu His Cys  Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 84)Asn Ser Ser Asn Tyr Cys Cys Glu Ile Cys  Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 85)Asn Ser Ser Asn Tyr Cys Cys Glu Lys Cys  Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 86)Asn Ser Ser Asn Tyr Cys Cys Glu Met Cys  Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 87)Asn Ser Ser Asn Tyr Cys Cys Glu Phe Cys  Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 88)Asn Ser Ser Asn Tyr Cys Cys Glu Pro Cys  Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 89)Asn Ser Ser Asn Tyr Cys Cys Glu Ser Cys  Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 90)Asn Ser Ser Asn Tyr Cys Cys Glu Thr Cys  Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 91Asn Ser Ser Asn Tyr Cys Cys Glu Trp Cys  Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 92)Asn Ser Ser Asn Tyr Cys Cys Glu Val Cys  Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 93)Cys Cys Glu Ala Cys Cys Asn Pro Ala Cys   Thr Gly Cys Tyr(SEQ ID NO: 94) Cys Cys Glu Arg Cys Cys Asn Pro Ala Cys  Thr Gly Cys Tyr (SEQ ID NO: 95)Cys Cys Glu Asn Cys Cys Asn Pro Ala Cys   Thr Gly Cys Tyr(SEQ ID NO: 96) Cys Cys Glu Asp Cys Cys Asn Pro Ala Cys  Thr Gly Cys Tyr (SEQ ID NO: 97)Cys Cys Glu Cys Cys Cys Asn Pro Ala Cys   Thr Gly Cys Tyr(SEQ ID NO: 98) Cys Cys Glu Gln Cys Cys Asn Pro Ala Cys  Thr Gly Cys Tyr (SEQ ID NO: 99)Cys Cys Glu Glu Cys Cys Asn Pro Ala Cys   Thr Gly Cys Tyr(SEQ ID NO: 100) Cys Cys Glu Gly Cys Cys Asn Pro Ala Cys  Thr Gly Cys Tyr (SEQ ID NO: 101)Cys Cys Glu His Cys Cys Asn Pro Ala Cys   Thr Gly Cys Tyr(SEQ ID NO: 102) Cys Cys Glu Ile Cys Cys Asn Pro Ala Cys  Thr Gly Cys Tyr (SEQ ID NO: 103)Cys Cys Glu Lys Cys Cys Asn Pro Ala Cys   Thr Gly Cys Tyr(SEQ ID NO: 104) Cys Cys Glu Met Cys Cys Asn Pro Ala Cys  Thr Gly Cys Tyr (SEQ ID NO: 105)Cys Cys Glu Phe Cys Cys Asn Pro Ala Cys   Thr Gly Cys Tyr(SEQ ID NO: 106) Cys Cys Glu Pro Cys Cys Asn Pro Ala Cys  Thr Gly Cys Tyr (SEQ ID NO: 107)Cys Cys Glu Ser Cys Cys Asn Pro Ala Cys   Thr Gly Cys Tyr(SEQ ID NO: 108) Cys Cys Glu Thr Cys Cys Asn Pro Ala Cys  Thr Gly Cys Tyr (SEQ ID NO: 109)Cys Cys Glu Trp Cys Cys Asn Pro Ala Cys   Thr Gly Cys Tyr(SEQ ID NO: 110) Cys Cys Glu Val Cys Cys Asn Pro Ala Cys  Thr Gly Cys Tyr (SEQ ID NO: 125)Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys   Thr Gly Cys (SEQ ID NO: 126)Cys Cys Glu Ala Cys Cys Asn Pro Ala Cys   Thr Gly Cys (SEQ ID NO: 127)Cys Cys Glu Arg Cys Cys Asn Pro Ala Cys   Thr Gly Cys (SEQ ID NO: 128)Cys Cys Glu Asn Cys Cys Asn Pro Ala Cys   Thr Gly Cys (SEQ ID NO: 129)Cys Cys Glu Asp Cys Cys Asn Pro Ala Cys   Thr Gly Cys (SEQ ID NO: 130)Cys Cys Glu Cys Cys Cys Asn Pro Ala Cys   Thr Gly Cys (SEQ ID NO: 131)Cys Cys Glu Gln Cys Cys Asn Pro Ala Cys   Thr Gly Cys (SEQ ID NO: 132)Cys Cys Glu Glu Cys Cys Asn Pro Ala Cys   Thr Gly Cys (SEQ ID NO: 133)Cys Cys Glu Gly Cys Cys Asn Pro Ala Cys   Thr Gly Cys (SEQ ID NO: 134)Cys Cys Glu His Cys Cys Asn Pro Ala Cys   Thr Gly Cys (SEQ ID NO: 135)Cys Cys Glu Ile Cys Cys Asn Pro Ala Cys   Thr Gly Cys (SEQ ID NO: 136)Cys Cys Glu Lys Cys Cys Asn Pro Ala Cys   Thr Gly Cys (SEQ ID NO: 137)Cys Cys Glu Met Cys Cys Asn Pro Ala Cys   Thr Gly Cys (SEQ ID NO: 138)Cys Cys Glu Phe Cys Cys Asn Pro Ala Cys   Thr Gly Cys (SEQ ID NO: 139)Cys Cys Glu Pro Cys Cys Asn Pro Ala Cys   Thr Gly Cys (SEQ ID NO: 140)Cys Cys Glu Ser Cys Cys Asn Pro Ala Cys   Thr Gly Cys (SEQ ID NO: 141)Cys Cys Glu Thr Cys Cys Asn Pro Ala Cys   Thr Gly Cys (SEQ ID NO: 142)Cys Cys Glu Trp Cys Cys Asn Pro Ala Cys   Thr Gly Cys (SEQ ID NO: 143)Cys Cys Glu Val Cys Cys Asn Pro Ala Cys   Thr Gly Cys

In an eighteenth aspect, the invention features a method for treatingcongestive heart failure, the method comprising administering a completeor partial agonist of the intestinal guanylate cyclase (GC-C) receptor.The agonist can be administered in combination with another agent fortreatment of congestive heart failure, for example, a natriureticpeptide such as atrial natriuretic peptide, brain natriuretic peptide orC-type natriuretic peptide), a diuretic, or an inhibitor of angiotensinconverting enzyme.

In a nineteenth aspect, the invention features a method for treatingBPH, the method comprising administering a complete or partial agonistof the intestinal guanylate cyclase (GC-C) receptor. The agonist can beadministered in combination with another agent for treatment of BPH, forexample, a 5-alpha reductase inhibitor (e.g., finasteride) or an alphaadrenergic inhibitor (e.g., doxazosine).

In a twentieth aspect, the invention features a method for treatingobesity, the method comprising administering a complete or partialagonist of the intestinal guanylate cyclase (GC-C) receptor. The agonistcan be administered in combination with another agent for treatment ofobesity, for example, gut hormone fragment peptide YY₃₋₃₆ (PYY₃₋₃₆) (N.Engl. J. Med. 349:941, 2003; ikpeapge daspeelnry yaslrhylnl vtrqry) or avariant thereof, glp-1 (glucagon-like peptide-1), exendin-4 (aninhibitor of glp-1), sibutramine, phentermine, phendimetrazine,benzphetamine hydrochloride (Didrex), orlistat (Xenical), diethylpropionhydrochloride (Tenuate), fluoxetine (Prozac), bupropion, ephedra,chromium, garcinia cambogia, benzocaine, bladderwrack (focusvesiculosus), chitosan, nomame herba, galega (Goat's Rue, French Lilac),conjugated linoleic acid, L-carnitine, fiber (psyllium, plantago, guarfiber), caffeine, dehydroepiandrosterone, germander (teucriumchamaedrys), B-hydroxy-β-methylbutyrate, ATL-962 (Alizyme PLC), andpyruvate. A peptide useful for treating obesity can be administered as aco-therapy with a peptide of the invention either as a distinct moleculeor as part of a fusion protein with a peptide of the invention. Thus,for example, PYY₃₋₃₆ can be fused to the carboxy or amino terminus of apeptide of the invention. Such a fusion protein can include achymotrypsin or trypsin cleavage site that can permit cleavage toseparate the two peptides. A peptide useful for treating obesity can beadministered as a co-therapy with electrostimulation (U.S. 20040015201).

In twenty first aspect, the invention features isolated nucleic acidmolecules comprising a sequence encoding a peptide of the invention andvectors, e.g., expression vectors that include such nucleic acidmolecules and can be used to express a peptide of the invention in acultured cell (e.g., a eukaryotice cell or a prokaryotic cell). Thevector can further include one or more regulatory elements, e.g., aheterologous promoter or elements required for translation operablylinked to the sequence encoding the peptide. In some cases the nucleicacid molecule will encode an amino acid sequence that includes the aminoacid sequence of a peptide of the invention. For example, the nucleicacid molecule can encode a preprotein or a preproprotein that can beprocessed to produce a peptide of the invention.

A vector that includes a nucleotide sequence encoding a peptide of theinvention or a peptide or polypeptide comprising a peptide of theinvention may be either RNA or DNA, single- or double-stranded,prokaryotic, eukaryotic, or viral. Vectors can include transposons,viral vectors, episomes, (e.g., plasmids), chromosomes inserts, andartificial chromosomes (e.g. BACs or YACs). Suitable bacterial hosts forexpression of the encode peptide or polypeptide include, but are notlimited to, E. coli. Suitable eukaryotic hosts include yeast such as S.cerevisiae, other fungi, vertebrate cells, invertebrate cells (e.g.,insect cells), plant cells, human cells, human tissue cells, and wholeeukaryotic organisms. (e.g., a transgenic plant or a transgenic animal).Further, the vector nucleic acid can be used to generate a virus such asvaccinia or baculovirus.

As noted above the invention includes vectors and genetic constructssuitable for production of a peptide of the invention or a peptide orpolypeptide comprising such a peptide. Generally, the genetic constructalso includes, in addition to the encoding nucleic acid molecule,elements that allow expression, such as a promoter and regulatorysequences. The expression vectors may contain transcriptional controlsequences that control transcriptional initiation, such as promoter,enhancer, operator, and repressor sequences. A variety oftranscriptional control sequences are well known to those in the art andmay be functional in, but are not limited to, a bacterium, yeast, plant,or animal cell. The expression vector can also include a translationregulatory sequence (e.g., an untranslated 5′ sequence, an untranslated3′ sequence, a poly A addition site, or an internal ribosome entrysite), a splicing sequence or splicing regulatory sequence, and atranscription termination sequence. The vector can be capable ofautonomous replication or it can integrate into host DNA.

The invention also includes isolated host cells harboring one of theforgoing nucleic acid molecules and methods for producing a peptide byculturing such a cell and recovering the peptide or a precursor of thepeptide. Recovery of the peptide or precursor may refer to collectingthe growth solution and need not involve additional steps ofpurification. Proteins of the present invention, however, can bepurified using standard purification techniques, such as, but notlimited to, affinity chromatography, thermaprecipitation, immunoaffinitychromatography, ammonium sulfate precipitation, ion exchangechromatography, filtration, electrophoresis and hydrophobic interactionchromatography.

In a twenty second aspect, the invention features a method of increasingthe level of cyclic guanosine 3′-monophosphate (cGMP) in an organ,tissue (e.g., the intestinal mucosa), or cell (e.g., a cell bearing GC-Areceptor) by administering a composition that includes a peptide of theinvention.

The peptides and agonist of the intestinal guanylate cyclase (GC-C)receptor can be used to treat constipation or decreased intestinalmotility, slow digestion or slow stomach emptying. The peptides can beused to relieve one or more symptoms of IBS (bloating, pain,constipation), GERD (acid reflux into the esophagus), functionaldyspepsia, or gastroparesis (nausea, vomiting, bloating, delayed gastricemptying) and other disorders described herein.

The details of one or more embodiments of the invention are set forth inthe accompanying description. All of the publications, patents andpatent applications are hereby incorporated by reference.

FIGURES

FIG. 1 depicts the results of LCMS analysis of recombinant MM-416776peptide and MD-915 peptide.

FIGS. 1 b and c depict the results of LCMS analysis of synthetic MD-1100peptide and the blank.

FIG. 2 depicts the results of the intestinal GC-C receptor activityassay of synthetic MM-416776 peptide, MD-915 peptide and two differentMD-1100 peptides.

FIG. 3 a depicts the effect of recombinant MM-416776 peptide andZelnorm® in an acute murine gastrointestinal transit model.

FIG. 3 b depicts the effect of synthetic MD-1100 peptide and Zelnorm® inan acute murine gastrointestinal transit model.

FIGS. 4 a and 4 b depict the effect of peptides MD-915, MD-1100, andMM-416776 in an acute murine gastrointestinal transit model.

FIG. 4 c depicts the effect of MD-1100 peptide in a chronic murinegastrointestinal transit model.

FIG. 5 a depicts the effect of MM-416776 peptide and Zelnorm® in asuckling mouse intestinal secretion model.

FIG. 5 b depicts the effects of MD-1100 and Zelnorm® in a mouseintestinal secretion model.

FIGS. 6 a and 6 b depict the effects of MM 416776, MD-1100 and MD-915peptides in a mouse intestinal secretion model.

FIG. 7 shows the results of experiment in which MD-1100 activity wasanalyzed in the TNBS colonic distention model.

FIGS. 8 a and 8 b show the effects of differing doses of MD-915 andMD-1100 in the PBQ writhing assay.

FIG. 9 shows the results of Kd determination analysis using MD-1100 in acompetitive radioligand binding assay.

FIGS. 10 a and 10 b show bioavailability data for IV and orallyadministered MD-1100 as detected by an ELISA assay and LCMS.

DETAILED DESCRIPTION

The peptides of the invention bind to the intestinal guanylate cyclase(GC-C) receptor, a key regulator of fluid and electrolyte balance in theintestine. When stimulated, this receptor, which is located on theapical membrane of the intestinal epithelial surface, causes an increasein intestinal epithelial cyclic GMP (cGMP). This increase in cGMP isbelieved to cause a decrease in water and sodium absorption and anincrease in chloride and potassium ion secretion, leading to changes inintestinal fluid and electrolyte transport and increased intestinalmotility. The intestinal GC-C receptor possesses an extracellular ligandbinding region, a transmembrane region, an intracellular proteinkinase-like region and a cyclase catalytic domain. Proposed functionsfor the GC-C receptor are fluid and electrolyte homeostasis, theregulation of epithelial cell proliferation and the induction ofapoptosis (Shalubhai 2002 Curr Opin Drug Dis Devel 5:261-268).

In addition to being expressed in the intestine by gastrointestinalepithelial cells, GC-C is expressed in extra-intestinal tissuesincluding kidney, lung, pancreas, pituitary, adrenal, developing liverand gall bladder (reviewed in Vaandrager 2002 Mol Cell Biochem230:73-83, Kulaksiz et al. 2004, Gastroenterology 126:732-740) and maleand female reproductive tissues (reviewed in Vaandrager 2002 Mol CellBiochem 230:73-83)) This suggests that the GC-C receptor agonists can beused in the treatment of disorders outside the GI tract, for example,congestive heart failure and benign prostatic hyperplasia.

Ghrelin, a peptide hormone secreted by the stomach, is a key regulatorof appetite in humans. Ghrelin expression levels are regulated byfasting and by gastric emptying (Kim et al. 2003 Neuroreprt 14:1317-20;Gualillo et al. 2003 FEBS Letts 552: 105-9). Thus, by increasinggastrointestinal motility, GC-C receptor agonists may also be used toregulate obesity.

In humans, the GC-C receptor is activated by guanylin (Gn) (U.S. Pat.No. 5,96,097), uroguanylin (Ugn) (U.S. Pat. No. 5,140,102) andlymphoguanylin (Forte et al. 1999 Endocrinology 140:1800-1806).Interestingly, these agents are 10-100 fold less potent than a class ofbacterially derived peptides, termed ST (reviewed in Gianella 1995 J LabClin Med 125:173-181). ST peptides are considered super agonists of GC-Cand are very resistant to proteolytic degradation.

ST peptide is capable of stimulating the enteric nervous system (Rolfeet al., 1994, J Physiolo 475: 531-537; Rolfe et al. 1999 Gut 44:615-619; Nzegwu et al. 1996 Exp Physiol 81: 313-315). Also, cGMP hasbeen reported to have anitnociceptive effects in multiple animal modelsof pain (Lazaro Ibanez et al. 2001 Eur J Pharmacol 426: 39-44; Soares etal. 2001 British J Pharmacol 134: 127-131; Jain et al. 2001 Brain Res909:170-178; Amarante et al. 2002 Eur J Pharmacol 454:19-23). Thus, GC-Cagonists may have both an analgesic as well an anti-inflammatory effect.

In bacteria, ST peptides are derived from a preproprotein that generallyhas at least 70 amino acids. The pre and pro regions are cleaved as partof the secretion process, and the resulting mature protein, whichgenerally includes fewer than 20 amino acids, is biologically active.

Among the known bacterial ST peptides are: E. coli ST Ib (Moseley et al.1983 Infect. Immun. 39:1167) having the mature amino acid sequence AsnSer Ser Asn Tyr Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr(SEQ ID NO:1); E. coli ST Ia (So and McCarthy 1980 Proc. Natl. Acad.Sci. USA 77:4011) having the mature amino acid sequence Asn Thr Phe TyrCys Cys Glu Leu Cys Cys Asn Pro Ala Cys Ala Gly Cys Tyr (SEQ ID NO:2);E. coli ST I* (Chan and Giannella 1981 J. Biol. Chem. 256:7744) havingthe mature amino acid sequence Asn Thr Phe Tyr Cys Cys Glu Leu Cys CysTyr Pro Ala Cys Ala Gly Cys Asn (SEQ ID NO:3); C. freundii ST peptide(Guarino et al. 1989b Infect. Immun. 57:649) having the mature aminoacid sequence Asn Thr Phe Tyr Cys Cys Glu Leu Cys Cys Asn Pro Ala CysAla Gly Cys Tyr (SEQ ID NO:4); Y. enterocolitica ST peptides,Y-ST(Y-STa), Y-STb, and Y-STc (reviewed in Huang et al. 1997 Microb.Pathog. 22:89) having the following pro-form amino acid sequences: GlnAla Cys Asp Pro Pro Ser Pro Pro Ala Glu Val Ser Ser Asp Trp Asp Cys CysAsp Val Cys Cys Asn Pro Ala Cys Ala Gly Cys (SEQ ID NO:5) (as well as aSer-7 to Leu-7 variant of Y-STa (SEQ ID NO:122), (Takao et al. 1985 Eur.J. Biochem. 152:199)); Lys Ala Cys Asp Thr Gln Thr Pro Ser Pro Ser GluGlu Asn Asp Asp Trp Cys Cys Glu Val Cys Cys Asn Pro Ala Cys Ala Gly Cys(SEQ ID NO:6); Gln Glu Thr Ala Ser Gly Gln Val Gly Asp Val Ser Ser SerThr Ile Ala Thr Glu Val Ser Glu Ala Glu Cys Gly Thr Gln Ser Ala Thr ThrGln Gly Glu Asn Asp Trp Asp Trp Cys Cys Glu Leu Cys Cys Asn Pro Ala CysPhe Gly Cys (SEQ ID NO:7), respectively; Y. kristensenii ST peptidehaving the mature amino acid sequence Ser Asp Trp Cys Cys Glu Val CysCys Asn Pro Ala Cys Ala Gly Cys (SEQ ID NO:8); V. cholerae non-01 STpeptide (Takao et al. (1985) FEBS lett. 193:250) having the mature aminoacid sequence Ile Asp Cys Cys Glu Ile Cys Cys Asn Pro Ala Cys Phe GlyCys Leu Asn (SEQ ID NO:9); and V. mimicus ST peptide (Arita et al. 1991FEMS Microbiol. Lett. 79:105) having the mature amino acid sequence IleAsp Cys Cys Glu Ile Cys Cys Asn Pro Ala Cys Phe Gly Cys Leu Asn (SEQ IDNO:10). Table I below provides sequences of all or a portion of a numberof mature ST peptides.

TABLE I GenBank ® GenBank ® Accession No. GI No. Sequence QHECIB   69638 NSSNYCCELCCNPACTGCY (SEQ ID NO: 1) P01559   123711NTFYCCELCCNPACAGCY (SEQ ID NO: 2) AAA24653   147878 NTFYCCELCCNPACAPCY(SEQ ID NO: 11) P01560   123707 NTFYCCELCCYPACAGCN (SEQ ID NO: 3)AAA27561   295439 IDCCEICCNPACFGCLN (SEQ ID NO: 9) P04429   123712IDCCEICCNPACFGCLN (SEQ ID NO: 10) S34671   421286 IDCCEICCNPACF(SEQ ID NO: 12) CAA52209   395161 IDCCEICCNPACFG (SEQ ID NO: 13) A54534  628844 IDCCEICCNPACFGCLN (SEQ ID NO: 14) AAL02159 15592919IDRCEICCNPACFGCLN (SEQ ID NO: 15) AAA18472   487395 DWDCCDVCCNPACAGC(SEQ ID NO: 16) S25659   282047 DWDCCDVCCNPACAGC (SEQ ID NO: 17) P74977 3913874 NDDWCCEVCCNPACAGC (SEQ ID NO: 18) BAA23656  2662339WDWCCELCCNPACFGC (SEQ ID NO: 19) P31518   399947 SDWCCEVCCNPACAGC(SEQ ID NO: 8)

The immature (including pre and pro regions) form of E. coli ST-1A(ST-P) protein has the sequence:mkklmlaifisvlsfpsfsqstesldsskekitletkkcdvvknnsekksenmnntfyccelccnpacagcy(SEQ ID NO:20; see GenBank® Accession No. P01559 (gi:123711). The presequence extends from aa 1-19. The pro sequence extends from aa 20-54.The mature protein extends from 55-72. The immature (including pre andpro regions) form of E. coli ST-1B (ST-H) protein has the sequence:mkksilfiflsvlsfspfaqdakpvesskekitleskkcniakksnksgpesmnssnyccelccnpactgcy(SEQ ID NO:21; see GenBank® Accession No. P07965 (gi:3915589). Theimmature (including pre and pro regions) form of Y. enterocolitica STprotein has the sequence:mkkivfvlvlmlssfgafgqetvsgqfsdalstpitaevykqacdpplppaevssdwdecdvccnpacagc(SEQ ID NO:22); see GenBank® Accession No. 525659 (gi:282047).

The peptides of the invention, like the bacterial ST peptides, have sixCys residues. These six Cys residues form three disulfide bonds in themature and active form of the peptide. If the six Cys residues areidentified, from the amino to carboxy terminus of the peptide, as A, B,C, D, E, and F, then the disulfide bonds form as follows: A-D, B-E, andC-F. The formation of these bonds is thought to be important for GC-Creceptor binding. Certain of the peptides of the invention include apotentially functional chymotrypsin cleavage site, e.g., a Trp, Tyr orPhe located between either Cys B and Cys D or between Cys E and Cys F.Cleavage at either chymotrypsin cleavage site reduces or eliminates theability of the peptide to bind to the GC-C receptor.

In the human body an inactive form of chymotrypsin, chymotrypsinogen isproduced in the pancreas. When this inactive enzyme reaches the smallintestine it is converted to active chymotrypsin by the excision of twodi-peptides. Active chymotrypsin can potentially cleave peptides at thepeptide bond on the carboxy-terminal side of Trp, Tyr or Phe. Thepresence of active chymotrypsin in the intestinal tract can potentiallylead to cleavage of certain of the peptides of the invention having anappropriately positioned functional chymotrypsin cleavage site. It isexpected that chymotrypsin cleavage will moderate the action of apeptide of the invention having an appropriately positioned chymotrypsincleavage site as the peptide passes through the intestinal tract.

Trypsinogen, like chymotrypsin, is a serine protease that is produced inthe pancreas and is present in the digestive tract. The active form,trypsin, will cleave peptides having a Lys or Arg. The presence ofactive trypsin in the intestinal tract can lead to cleavage of certainof the peptides of the invention having an appropriately positionedfunctional trypsin cleavage site. It is expected that chymotrypsincleavage will moderate the action of a peptide of the invention havingan appropriately positioned trypsin cleavage site as the peptide passesthrough the intestinal tract.

Many gastrointestinal disorders, including IBS, are associated withabdominal or visceral pain. Certain of the peptides of the inventioninclude analgesic or antinociceptive tags such as the carboxy-terminalsequence AspPhe immediately following a Trp, Tyr or Phe that creates afunctional chymotrypsin cleavage site or following Lys or Arg thatcreates a functional trypsin cleavage site. Chymotrypsin in theintestinal tract can potentially cleave such peptides immediatelycarboxy terminal to the Trp, Phe or Tyr residue, releasing thedipeptide, AspPhe. This dipeptide has been shown to have analgesicactivity in animal models (Abdikkahi et al. 2001 Fundam Clin Pharmacol15:117-23; Nikfar et al 1997, 29:583-6; Edmundson et al 1998 ClinPharmacol Ther 63:580-93). In this manner such peptides can treat bothpain and inflammation. Other analgesic peptides can be present at thecarboxy terminus of the peptide (following a functional cleavage site)including: endomorphin-1, endomorphin-2, nocistatin, dalargin, lupron,and substance P.

A number of the useful peptides are based on the core sequence: Cys CysGlu Leu Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO:29). Tocreate a variant having a potentially functional chymotrypsin cleavagesite capable of inactivating the peptide, either the Leu (underlined) orthe Thr (underlined) can be replaced by Trp, Phe or Tyr or both the Leuand the Thr can be replaced by (independently) Trp, Phe or Tyr. Tocreate a variant having an analgesic di-peptide, the core sequence isfollowed by Asp Phe. The carboxy terminal Tyr in the core sequence canallow the Asp Phe dipeptide to be released by chymotrypsin in thedigestive tract. The core sequence can be optionally be preceded by AsnSer Ser Asn Tyr or Asn.

Thus, useful variants based on the core sequence include:

(SEQ ID NO: 26; MM-416776) Asn Ser Ser Asn Tyr Cys Cys Glu Leu Cys  Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 27)Asn Ser Ser Asn Tyr Cys Cys Glu Leu Cys  Cys Asn Pro Ala Cys Trp Gly Cys Tyr (SEQ ID NO: 28; MD-915)Asn Ser Ser Asn Tyr Cys Cys Glu Tyr Cys  Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ ID NO: 29; MM416774)Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys   Thr Gly Cys Tyr(SEQ ID NO: 30) Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys  Trp Gly Cys Tyr (SEQ ID NO: 31; MD-1100)Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys   Thr Gly Cys Tyr(SEQ ID NO: 32) Asn Cys Cys Glu Leu Cys Cys Asn Pro Ala  Cys Thr Gly Cys Tyr (SEQ ID NO: 33)Asn Cys Cys Glu Leu Cys Cys Asn Pro Ala   Cys Trp Gly Cys Tyr(SEQ ID NO: 34) Asn Cys Cys Glu Phe Cys Cys Asn Pro Ala  Cys Thr Gly Cys Tyr (SEQ ID NO: 35)Asn Cys Cys Glu Tyr Cys Cys Asn Pro Ala   Cys Thr Gly Cys Tyr(SEQ ID NO: 36) Asn Cys Cys Glu Trp Cys Cys Asn Pro Ala  Cys Thr Gly Cys Tyr (SEQ ID NO: 37)Asn Cys Cys Glu Arg Cys Cys Asn Pro Ala   Cys Thr Gly Cys Tyr(SEQ ID NO: 38) Asn Cys Cys Glu Lys Cys Cys Asn Pro Ala  Cys Thr Gly Cys Tyr (SEQ ID NO: 39)Asn Ser Ser Asn Tyr Cys Cys Glu Leu Cys  Cys Asn Pro Ala Cys Thr Gly Cys Tyr Asp  Phe (SEQ ID NO: 40)Asn Ser Ser Asn Tyr Cys Cys Glu Leu Cys  Cys Asn Pro Ala Cys Trp Gly Cys Tyr Asp  Phe (SEQ ID NO: 41)Asn Ser Ser Asn Tyr Cys Cys Glu Phe Cys  Cys Asn Pro Ala Cys Thr Gly Cys Tyr Asp  Phe (SEQ ID NO: 42)Asn Ser Ser Asn Tyr Cys Cys Glu Tyr Cys  Cys Asn Pro Ala Cys Thr Gly Cys Tyr Asp  Phe (SEQ ID NO: 43)Asn Ser Ser Asn Tyr Cys Cys Glu Trp Cys  Cys Asn Pro Ala Cys Thr Gly Cys Tyr Asp  Phe (SEQ ID NO: 44)Asn Ser Ser Asn Tyr Cys Cys Glu Arg Cys  Cys Asn Pro Ala Cys Thr Gly Cys Tyr Asp  Phe (SEQ ID NO: 45)Asn Ser Ser Asn Tyr Cys Cys Glu Lys Cys  Cys Asn Pro Ala Cys Thr Gly Cys Tyr Asp  Phe (SEQ ID NO: 46)Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys   Thr Gly Cys Tyr Asp Phe(SEQ ID NO: 47) Cys Cys Glu Leu Cys Cys Asn Pro Ala Cys  Trp Gly Cys Tyr Asp Phe (SEQ ID NO: 48)Cys Cys Glu Phe Cys Cys Asn Pro Ala Cys   Thr Gly Cys Tyr Asp Phe(SEQ ID NO: 49) Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys  Thr Gly Cys Tyr Asp Phe (SEQ ID NO: 50)Cys Cys Glu Trp Cys Cys Asn Pro Ala Cys   Thr Gly Cys Tyr Asp Phe(SEQ ID NO: 51) Cys Cys Glu Arg Cys Cys Asn Pro Ala Cys  Thr Gly Cys Tyr Asp Phe (SEQ ID NO: 52)Cys Cys Glu Lys Cys Cys Asn Pro Ala Cys   Thr Gly Cys Tyr Asp Phe(SEQ ID NO: 53) Asn Cys Cys Glu Leu Cys Cys Asn Pro Ala  Cys Thr Gly Cys Tyr Asp Phe (SEQ ID NO: 54)Asn Cys Cys Glu Leu Cys Cys Asn Pro Ala   Cys Trp Gly Cys Tyr Asp Phe(SEQ ID NO: 55) Asn Cys Cys Glu Phe Cys Cys Asn Pro Ala  Cys Thr Gly Cys Tyr Asp Phe (SEQ ID NO: 56)Asn Cys Cys Glu Tyr Cys Cys Asn Pro Ala   Cys Thr Gly Cys Tyr Asp Phe(SEQ ID NO: 57) Asn Cys Cys Glu Trp Cys Cys Asn Pro Ala  Cys Thr Gly Cys Tyr Asp Phe (SEQ ID NO: 58)Asn Cys Cys Glu Arg Cys Cys Asn Pro Ala   Cys Thr Gly Cys Tyr Asp Phe(SEQ ID NO: 59) Asn Cys Cys Glu Lys Cys Cys Asn Pro Ala  Cys Thr Gly Cys Tyr Asp Phe

In some cases, the peptides of the invention are produced as a preproprotein that includes the amino terminal leader sequence:mkksilfiflsvlsfspfaqdakpvesskekitleskkcniakksnksgpesmn. Where thepeptide is produced by a bacterial cell, e.g., E. coli, the forgoingleader sequence will be cleaved and the mature peptide will beefficiently secreted from the bacterial cell. U.S. Pat. No. 5,395,490describes vectors, expression systems and methods for the efficientproduction of ST peptides in bacterial cells and methods for achievingefficient secretion of mature ST peptides. The vectors, expressionsystems and methods described in U.S. Pat. No. 5,395,490 can be used toproduce the ST peptides and variant ST peptides of the present invention

Variant Peptides

The invention includes variant peptides which can include one, two,three, four, five, six, seven, eight, nine, or ten (in some embodimentsfewer than 5 or fewer than 3 or 2 or fewer) amino acid substitutionsand/or deletions compared to SEQ ID NOs:26 to 59, 66 to 110 and 125 to143. The substitution(s) can be conservative or non-conservative. Thenaturally-occurring amino acids can be substituted by D-isomers of anyamino acid, non-natural amino acids, and other groups. A conservativeamino acid substitution results in the alteration of an amino acid for asimilar acting amino acid, or amino acid of like charge, polarity, orhydrophobicity. At some positions, even conservative amino acidsubstitutions can reduce the activity of the peptide. A conservativesubstitution can substitute a naturally-occurring amino acid for anon-naturally-occurring amino acid. The amino acid substitutions amongnaturally-occurring amino acids are listed in Table II

TABLE II For Amino Acid Code Replace with any of Alanine Ala Gly, Cys,Ser Arginine Arg Lys, His Asparagine Asn Asp, Glu, Gln, Aspartic AcidAsp Asn, Glu, Gln Cysteine Cys Met, Thr, Ser Glutamine Gln Asn, Glu, AspGlutamic Acid Glu Asp, Asn, Gln Glycine Gly Ala Histidine His Lys, ArgIsoleucine Ile Val, Leu, Met Leucine Leu Val, Ile, Met Lysine Lys Arg,His Methionine Met Ile, Leu, Val Phenylalanine Phe Tyr, His, Trp ProlinePro Serine Ser Thr, Cys, Ala Threonine Thr Ser, Met, Val Tryptophan TrpPhe, Tyr Tyrosine Tyr Phe, His Valine Val Leu, Ile, Met

In some circumstances it can be desirable to treat patients with avariant peptide that binds to and activates intestinal GC-C receptor,but is less active than the non-variant form the peptide. This reducedactivity can arise from reduced affinity for the receptor or a reducedability to activate the receptor once bound or reduced stability of thepeptide. In some peptides pairs of Cys residues which normally form adisulfide bond one or both members of the pair can be replaced byhomocysteine, 3-mercaptoproline (Kolodziej et al. 1996 Int J PeptProtein Res 48:274); β, β dimethylcysteine (Hunt et al. 1993 Int J PeptProtein Res 42:249) or diaminopropionic acid (Smith et al. 1978 J MedChem 21:117) to form alternative internal cross-links at the positionsof the normal disulfide bonds.

Production of Peptides

Useful peptides can be produced either in bacteria including, withoutlimitation, E. coli, or in other existing systems for peptide or proteinproduction (e.g., Bacillus subtilis, baculovirus expression systemsusing Drosophila Sf9 cells, yeast or filamentous fungal expressionsystems, mammalian cell expression systems), or they can be chemicallysynthesized.

If the peptide or variant peptide is to be produced in bacteria, e.g.,E. coli, the nucleic acid molecule encoding the peptide will preferablyalso encode a leader sequence that permits the secretion of the maturepeptide from the cell. Thus, the sequence encoding the peptide caninclude the pre sequence and the pro sequence of, for example, anaturally-occurring bacterial ST peptide. The secreted, mature peptidecan be purified from the culture medium.

The sequence encoding a peptide of the invention is preferably insertedinto a vector capable of delivering and maintaining the nucleic acidmolecule in a bacterial cell. The DNA molecule may be inserted into anautonomously replicating vector (suitable vectors include, for example,pGEM3Z and pcDNA3, and derivatives thereof). The vector nucleic acid maybe a bacterial or bacteriophage DNA such as bacteriophage lambda or M13and derivatives thereof. Construction of a vector containing a nucleicacid described herein can be followed by transformation of a host cellsuch as a bacterium. Suitable bacterial hosts include but are notlimited to, E. coli, B. subtilis, Pseudomonas, Salmonella. The geneticconstruct also includes, in addition to the encoding nucleic acidmolecule, elements that allow expression, such as a promoter andregulatory sequences. The expression vectors may contain transcriptionalcontrol sequences that control transcriptional initiation, such aspromoter, enhancer, operator, and repressor sequences. A variety oftranscriptional control sequences are well known to those in the art.The expression vector can also include a translation regulatory sequence(e.g., an untranslated 5′ sequence, an untranslated 3′ sequence, or aninternal ribosome entry site). The vector can be capable of autonomousreplication or it can integrate into host DNA to ensure stability duringpeptide production.

The protein coding sequence that includes a peptide of the invention canalso be fused to a nucleic acid encoding a polypeptide affinity tag,e.g., glutathione S-transferase (GST), maltose E binding protein,protein A, FLAG tag, hexa-histidine, myc tag or the influenza HA tag, inorder to facilitate purification. The affinity tag or reporter fusionjoins the reading frame of the peptide of interest to the reading frameof the gene encoding the affinity tag such that a translational fusionis generated. Expression of the fusion gene results in translation of asingle polypeptide that includes both the peptide of interest and theaffinity tag. In some instances where affinity tags are utilized, DNAsequence encoding a protease recognition site will be fused between thereading frames for the affinity tag and the peptide of interest.

Genetic constructs and methods suitable for production of immature andmature forms of the peptides and variants of the invention in proteinexpression systems other than bacteria, and well known to those skilledin the art, can also be used to produce peptides in a biological system.

Mature peptides and variants thereof can be synthesized by thesolid-phase method using an automated peptide synthesizer. For example,the peptide can be synthesized on Cyc(4-CH₂Bxl)-OCH₂-4-(oxymethyl)-phenylacetamidomethyl resin using a doublecoupling program. Protecting groups must be used appropriately to createthe correct disulfide bond pattern. For example, the followingprotecting groups can be used: t-butyloxycarbonyl (alpha-amino groups);acetamidomethyl (thiol groups of Cys residues B and E); 4-methylbenzyl(thiol groups of Cys residues C and F); benzyl (y-carboxyl of glutamicacid and the hydroxyl group of threonine, if present); and bromobenzyl(phenolic group of tyrosine, if present). Coupling is effected withsymmetrical anhydride of t-butoxylcarbonylamino acids orhydroxybenzotriazole ester (for asparagine or glutamine residues), andthe peptide is deprotected and cleaved from the solid support inhydrogen fluoride, dimethyl sulfide, anisole, and p-thiocresol using8/1/1/0.5 ratio (v/v/v/w) at 0° C. for 60 min. After removal of hydrogenfluoride and dimethyl sulfide by reduced pressure and anisole andp-thiocresol by extraction with ethyl ether and ethyl acetatesequentially, crude peptides are extracted with a mixture of 0.5M sodiumphosphate buffer, pH 8.0 and N,N-dimethylformamide using 1/1 ratio, v/v.The disulfide bond for Cys residues B and E is the formed using dimethylsulfoxide (Tam et al. (1991) J. Am. Chem. Soc. 113:6657-62). Theresulting peptide is the purified by reverse-phase chromatography. Thedisulfide bond between Cys residues C and F is formed by firstdissolving the peptide in 50% acetic acid in water. Saturated iodinesolution in glacial acetic acid is added (1 ml iodine solution per 100ml solution). After incubation at room temperature for 2 days in anenclosed glass container, the solution is diluted five-fold withdeionized water and extracted with ethyl ether four times for removal ofunreacted iodine. After removal of the residual amount of ethyl ether byrotary evaporation the solution of crude product is lyophilized andpurified by successive reverse-phase chromatography.

Intestinal GC-C Receptor Binding Assay

The ability of peptides and other agents to bind to the intestinal GC-Creceptor can be tested as follows. Cells of the T84 human coloncarcinoma cell line (American Type Culture Collection (Bethesda, Md.)are grown to confluence in 24-well culture plates with a 1:1 mixture ofHam's F12 medium and Dulbecco's modified Eagle's medium (DMEM),supplemented with 5% fetal calf serum. Cells used in the assay aretypically between passages 54-60. Briefly, T84 cell monolayers in24-well plates are washed twice with 1 ml of binding buffer (DMEMcontaining 0.05% bovine serum albumin and 25 mM HEPES, pH 7.2), thenincubated for 30 min at 37° C. in the presence of mature radioactivelylabeled E. coli ST peptide and the test material at variousconcentrations. The cells are then washed four times with 1 ml of DMEMand solubilized with 0.5 ml/well 1N NaOH. The level of radioactivity inthe solubilized material is then determined using standard methods.

Example 1 Preparation of Variant ST Peptides and Wild-Type ST Peptide1a: Preparation of Recombinant Variant ST Peptides and Wild-Type STPeptide

A variant ST peptide, referred to as MD-915, was reproducedrecombinantly and tested in an animal model. MD-915 has the sequence:Asn Ser Ser Asn Tyr Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly CysTyr (SEQ ID NO:28). A peptide having the sequence of the wild-type STpeptide was also created (MM-416776).

MD-915 and MM-416776 peptides were produced as preproproteins usingvectors produced as follows. A sequence encoding a heat-stableenterotoxin pre-pro sequence was amplified from pGK51/pGSK51 (ATCC67728) using oligonucleotide M03514 (5′CACACCATATGAAGAAATCAATATTATTTATTTTTCTTTCTG 3′ (SEG ID NO:60)) andoligonucleotide M03515 (5′CACACCTCGAGTTAGGTCTCCATGCTTTCAGGACCACTTTTATTAC3′ (SEQ ID NO: 61)). The amplification product fragment was digestedwith NdeI/XhoI and ligated to the T7 expression vector, pET26b(+)(Novagen) digested with NdeI/XhoI thereby creating plasmid MB3976. Theregion encoding the pre-pro protein was sequenced and found to encodethe amino acid sequence:mkksilfiflsvlsfspfaqdakpagsskekitleskkcnivkksnksgpesm (SEQ ID NO: 24)which differs from the amino acid sequence of heat-stable enterotoxin a2precursor (sta2; mkksilfiflsvlsfspfaqdakpagsskekitleskkcnivkknnesspesm(SEQ ID NO:25); GenBank® Accession No. Q47185, GI: 3913876) at threepositions (indicated by underlining and bold text) near the C-terminus.To create expression vectors with the pre-pro sequence, complementaryoligos encoding each ST peptide variant or wild-type ST peptide wereannealed and cloned into the MB3976 expression vector. To create MB3984(encoding MM-416776 peptide full length wild-type ST peptide as a preproprotein), containing the amino acid sequence, NSSNYCCELCCNPACTGCY (SEQID NO:26) fused downstream of the pre-pro sequence, MB 3976 was digestedwith BsaI/XhoI and ligated to annealed oligos MO3621(5′GCATGAATAGTAGCAATTACTGCTGTGAATTGTGTTGTAATCCTGCTTGTACCGGGTGCTATTAATAAC 3′ (SEQ ID NO:62)) and M03622 (5′TCGAGTTATTAATAGCACCCGGTACAAGCAGGATTACAACACAATTCACAGCAGTA ATTGCTACTATTC3′(SEQ ID NO:63)). To create MB3985 (encoding MD-915 as a preproprotein) containing the following amino acid sequence,NSSNYCCEYCCNPACTGCY (SEQ ID NO:28) fused downstream of the pre-prosequence, MB 3976 was digested with BsaI/XhoI and ligated to annealedoligos M03529 (5′GCATGAATAGTAGCAATTACTGCTGTGAATATTGTTGTAATCCTGCTTGTACCGGGT GCTATTAATAAC3′ (SEQ ID NO:64)) and M03530 (5′TCGAGTTATTAATAGCACCCGGTACAAGCAGGATTACAACAATATTCACAGCAGTA ATTGCTACTATTC3′(SEQ ID NO:65)).

The MD-915 peptide and the MM-416776 peptide were produced as follows.The expression vectors were transformed into E. coli bacterial hostBL21λ DE3 (Invitrogen). A single colony was innoculated and grownshaking overnight at 30° C. in L broth+25 mg/l kanamycin. The overnightculture was added to 3.2 L of batch medium (Glucose 25 g/l, CaseaminoAcids 5 g/l, Yeast Extract 5 g/l, KH₂PO₄ 13.3 (NH₄)₂HPO₄ 4 g/l,MgSO₄-7H₂0 1.2 g/l, Citric Acid 1.7 g/l, EDTA 8.4 mg/l, CoCl₂-6H₂O 2.5mg/l, MnCl₂-4H₂O 15 mg/l, CuCl₂-4H₂0 1.5 mg/l, H₃BO₃ 3 mg/l,Na₂MoO₄-2H₂0 2.5 mg/l, Zn Acetate-2H₂0 13 mg/l, Ferric Citrate 100 mg/l,Kanamycin 25 mg/l, Antifoam DF₂0₄ 1 ml/l) and fermented using thefollowing process parameters: pH 6.7—control with base only (28% NH₄OH),30° C., aeration: 5 liters per minute. After the initial consumption ofbatch glucose (based on monitoring dissolved oxygen (DO) levels), 1.5 Lof feed medium (Glucose 700 g/l, Caseamino Acids 10 g/l, Yeast Extract10 g/l, MgSO₄-7H₂0 4 g/l, EDTA 13 mg/l, CoCl₂-6H₂O 4 mg/l, MnCl₂-4H₂O23.5 mg/l, CuCl₂-4H₂0 2.5 mg/l, H₃BO₃ 5 mg/l, Na₂MoO₄-2H₂0 4 mg/l, ZnAcetate-2H₂0 16 mg/l, Ferric Citrate 40 mg/l, Antifoam DF₂0₄ 1 ml/l) wasadded at a feed rate controlled to maintain 20% DO. IPTG was added to0.2 mM 2 hours post feed start. The total run time was approximately40-45 hours (until feed exhaustion).

Cells were collected by centrifugation at 5,000 g for 10 minutes. Thecell pellet was discarded and the supernatant was passed through a 50 Kdultrafiltration unit. The 50 Kd filtrate (0.6 liters) was loaded onto a110 ml Q-Sepharose fast Flow column (Amersham Pharmacia, equilibratedwith 20 mM Tris-HCl pH 7.5) at a flow rate of 400 ml/hour. The columnwas washed with six volumes of 20 mM Tris-HCl pH 7.5 and proteins wereeluted with 50 mM acetic acid collecting 50 ml fractions. Fractionscontaining ST peptide variant or wild-type ST peptide were pooled andthe solvent was removed by rotary evaporation. The dried proteins wereresuspended in 10 ml of 8% acetic acid, 0.1% trifluoroacetic acid (TFA)and loaded onto a Varian Polaris C18-A column (250×21.2 mm 10 Tm,equilibrated in the same buffer) at a flow rate of 20 ml/min. The columnwas washed with 100 ml of 8% methanol, 0.1% TFA and developed with agradient (300 ml) of 24 to 48% methanol, 0.1% TFA, collecting 5-mlfractions. Fractions containing peptide were pooled and the solvent wasremoved by rotary evaporation. The peptides were dissolved in 0.1% TFAand lyophilized.

The MD-915 peptide and MM-416776 peptide fractions were analyzed bystandard LCMS and HPLC. LCMS analysis revealed that MD-915 is morehomogeneous than MM-416776 (see FIG. 1 a; note that MD-915 peptideexhibits fewer peaks (Panel B) than MM-416776 (Panel A)).

1b: Preparation of Synthetic Variant ST Peptides and Wild-Type STPeptide

Peptides were chemically synthesized by a commercial peptide synthesiscompany. Varying yields of peptides were obtained depending on theefficiency of chemical synthesis. Thus, the four peptides, in decreasingorder of yield were: Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly CysTyr (SEQ ID NO:31; MD-1100), 10-20% yield; Cys Cys Glu Leu Cys Cys AsnPro Ala Cys Thr Gly Cys Tyr (SEQ ID NO:29; MM416774); Asn Ser Ser AsnTyr Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys Tyr (SEQ IDNO:28; M1)-915); Asn Ser Ser Asn Tyr Cys Cys Glu Leu Cys Cys Asn Pro AlaCys Thr Gly Cys Tyr (SEQ ID NO:26 MM-416776), <5% yield. Thus thespecific amino acid changes introduced into the peptides can createimproved manufacturing properties.

FIG. 1 b shows the total ion chromatograph profile of syntheticallymanufactured MD-1100. FIG. 1 c shows the total ion chromatograph profileof the control blank sample. There is one major peak present in theMD-1100 sample that is not also present in the control sample.Quantitative analysis suggests the MD-1100 is >98% pure.

Example 2 Activation of the Intestinal GC-C Receptor by a Variant STPeptide and ST Peptide

The ability of MD-915, MM-416776, and MD-1100 to activate the intestinalGC-C receptor was assessed in an assay employing the T84 human coloncarcinoma cell line (American Type Culture Collection (Bethesda, Md.).For the assays cells were grown to confluency in 24-well culture plateswith a 1:1 mixture of Ham's F12 medium and Dulbecco's modified Eagle'smedium (DMEM), supplemented with 5% fetal calf serum and were used atbetween passages 54 and 60.

Briefly, monolayers of T84 cells in 24-well plates were washed twicewith 1 ml/well DMEM, then incubated at 37° C. for 10 min with 0.45 mlDMEM containing 1 mM isobutylmethylxanthine (IBMX), a cyclic nucleotidephosphodiesterase inhibitor. Test peptides (50 μl) were then added andincubated for 30 minutes at 37° C. The media was aspirated and thereaction was then terminated by the addition of ice cold 0.5 ml of 0.1NHCl. The samples were held on ice for 20 minutes and then evaporated todryness using a heat gun or vacuum centrifugation. The dried sampleswere resuspended in 0.5 ml of phosphate buffer provided in the CaymanChemical Cyclic GMP EIA kit (Cayman Chemical, Ann Arbor, Mich.). CyclicGMP was measured by EIA according to procedures outlined in the CaymanChemical Cyclic GMP EIA kit.

FIG. 2 shows the activity of chemically synthesized peptide variants inthis GC-C receptor activity assay. In this assay, MM-416776 and twodifferent MD-1100 peptides (MD-1100(a) and MD-1100(b), synthesized bytwo different methods) had activity comparable to MM-416776. MD-915 andMM-416776 peptide were chemically synthesized in a manner identical tothat of MD-1100(b).

Example 3 MD-915 and MM-416776 Increase Intestinal Transit in Mice

In order to determine whether the peptides increase the rate ofgastrointestinal transit, the peptides and controls were tested using amurine gastrointestinal transit (GIT) assay (Moon et al. Infection andImmunity 25:127, 1979). In this assay, charcoal, which can be readilyvisualized in the gastrointestinal tract is administered to mice afterthe administration of a test compound. The distance traveled by thecharcoal is measured and expressed as a percentage of the total lengthof the colon.

Mice were fasted with free access to water for 12 to 16 hours before thetreatment with peptide or control buffer. The peptides were orallyadministered at 1 μg/kg-1 mg/kg of peptide in buffer (20 mM Tris pH 7.5)7 minutes before being given an oral dose of 5% Activated Carbon(Aldrich 242276-250G). Control mice were administered buffer only beforebeing given a dose of Activated Carbon. After 15 minutes, the mice weresacrificed and their intestines from the stomach to the cecum weredissected. The total length of the intestine as well as the distancetraveled from the stomach to the charcoal front was measured for eachanimal and the results are expressed as the percent of the total lengthof the intestine traveled by the charcoal front. All results arereported as the average of 10 mice±standard deviation. A comparison ofthe distance traveled by the charcoal between the mice treated withpeptide versus the mice treated with vehicle alone was performed using aStudent's t test and a statistically significant difference wasconsidered for P<0.05. P-values are calculated using a two-sided T-Testassuming unequal variances.

As can be seen in FIG. 3 a, b, wild-type ST peptide (MM-416776,(Sigma-Aldrich, St Louis, Mo.; 0.1 mg/kg), synthetically manufacturedMD-1100 and Zelnorm® (0.1 mg/kg), a drug approved for IBS that is anagonist for the serotonin receptor 5HT4, increase gastrointestinaltransit rate in this model. FIG. 4 a shows the result of a studydemonstrating that intestinal transit rate increases with an increasingdosage of either recombinantly synthesized MM-416776 or MD-915. FIG. 4 bshows the results of a study demonstrating both chemically synthesizedMM-416776 or MD-1100 peptide increase intestinal transit rates more thaneither Tris buffer alone or an equivalent dose of Zelnorm®.

The identical experiment was performed to determine if MD-1100 iseffective in a chronic dosing treatment regimen. Briefly, 8 week old CD1female mice are dosed orally once a day for 5 days with either MID-1100(0.06 mg/kg or 0.25 mg/kg in 20 mM Tris pH 7.5) or vehicle alone (20 mMTris pH 7.5). On the 5^(th) day, a GIT assay is performed identical tothat above except 200 μl of a 10% charcoal solution is administered.FIG. 4 c shows the results of a study demonstrating both chemicallysynthesized MD-1100 or Zelnorm® are effective in a mousegastrointestinal motility assay upon chronic dosing (daily for 5 days).The results are shown side by side with acute dosing (1 day).

Example 4 MD-915 Peptide and MM-416776 Peptide Increase IntestinalSecretion in Suckling Mice (SuMi Assay)

MM-416776 peptide and MD-915 were tested for their ability to increaseintestinal secretion using a suckling mouse model of intestinalsecretion. In this model a test compound is administered to sucklingmice that are between 7 and 9 days old. After the mice are sacrificed,the gastrointestinal tract from the stomach to the cecum is dissected(“guts”). The remains (“carcass”) as well as the guts are weighed andthe ratio of guts to carcass weight is calculated. If the ratio is above0.09, one can conclude that the test compound increases intestinalsecretion. FIG. 5 a shows a dose response curve for wild-type ST peptide(MM-416776) in this model. FIG. 5 b shows dose response curve for theMD-1100 peptide in this model. These data show that wild-type ST peptide(purchased from TDT, Inc. West Chester, Pa.) and the MD-1100 peptideincrease intestinal secretion. The effect of Zelnorm® was also studied.As can be seen from FIG. 5, Zelnorm® at 0.2 mg/kg does not increaseintestinal secretion in this model. FIG. 6 a shows a dose response curvefor the recombinant MM-416776 peptide described above and therecombinant MD-915 peptide described above. As can be seen from FIG. 6a, both peptides increase intestinal secretion in this model. SimilarlyFIG. 6 b shows a dose response curve for chemically synthesized MD-915,MD-1100 and MM-416776 as well as wild-type ST peptide (purchased fromSigma-Aldrich, St Louis, Mo.).

Colonic Hyperalgesia Animal Models

Hypersensitivity to colorectal distension is common in patients with IBSand may be responsible for the major symptom of pain. Both inflammatoryand non-inflammatory animal models of visceral hyperalgesia todistension have been developed to investigate the effect of compounds onvisceral pain in IBS.

I. Trinitrobenzenesulphonic Acid (TNBS)-Induced Rectal Allodynia Model

Male Wistar rats (220-250 g) were premedicated with 0.5 mg/kg ofacepromazine injected intraperitoneally (IP) and anesthetized byintramuscular administration of 100 mg/kg of ketamine. Pairs of nichromewire electrodes (60 cm in length and 80 μm in diameter) were implantedin the striated muscle of the abdomen, 2 cm laterally from the whiteline. The free ends of electrodes were exteriorized on the back of theneck and protected by a plastic tube attached to the skin.Electromyographic (EMG) recordings were started 5 days after surgery.Electrical activity of abdominal striated muscle was recorded with anelectroencephalograph machine (Mini VIII, Alvar, Paris, France) using ashort time constant (0.03 sec.) to remove low-frequency signals (<3 Hz).

Ten days post surgical implantation, trinitrobenzenesulphonic acid(TNBS) was administered to induce rectal inflammation. TNBS (80 mg kg⁻¹in 0.3 ml 50% ethanol) was administered intrarectally through a siliconerubber catheter introduced at 3 cm from the anus under lightdiethyl-ether anesthesia, as described (Morteau et al. 1994 Dig Dis Sci39:1239). Following TNBS administration, rats were placed in plastictunnels where they were severely limited in mobility for several daysbefore colorectal distension (CRD). Experimental compound wasadministered one hour before CRD which was performed by insertion intothe rectum, at 1 cm of the anus, a 4 cm long balloon made from a latexcondom (Gue et al, 1997 Neurogastroenterol. Motil. 9:271). The balloonwas fixed on a rigid catheter taken from an embolectomy probe (Fogarty).The catheter attached balloon was fixed at the base of the tail. Theballoon, connected to a barostat, was inflated progressively by step of15 mmHg, from 0 to 60 mmHg, each step of inflation lasting 5 min.Evaluation of rectal sensitivity, as measured by EMG, was performedbefore (1-2 days) and 3 days following rectal instillation of TNBS.

The number of spike bursts that corresponds to abdominal contractionswas determined per 5 min periods. Statistical analysis of the number ofabdominal contractions and evaluation of the dose-effects relationshipswas performed by a one way analysis of variance (ANOVA) followed by apost-hoc (Student or Dunnett tests) and regression analysis for ED50 ifappropriate.

FIG. 7 shows the results of experiment in which MD-1100 activity wasanalyzed in the TNBS colorectal model. Significant decreases inabdominal response are observed at 0.3 μg/kg and 3 μg/kg MD-1100. Theseresults demonstrate that MD-1100 reduces pain associated with colorectaldistension in this animal model.

II. Stress-Induced Hyperalgesia Model

Male Wistar Rats (200-250 g) are surgically implanted with nichrome wireelectrodes as in the TNBS model. Ten days post surgical implantation,partial restraint stress (PRS), is performed as described by Williams etal. for two hours (Williams et al. 1988 Gastroenterology 64:611).Briefly, under light anesthesia with ethyl-ether, the foreshoulders,upper forelimbs and thoracic trunk are wrapped in a confining harness ofpaper tape to restrict, but not prevent body movements. Controlsham-stress animals are anaesthetized but not wrapped. Thirty minutesbefore the end of the PRS session, the animals are administeredtest-compound or vehicle. Thirty minutes to one hour after PRScompletion, the CRD distension procedure is performed as described abovefor the TNBS model with barostat at pressures of 15, 30, 45 and 60 mmHg. Statistical analysis on the number of bursts is determined andanalyzed as in the TNBS model above.

Phenylbenzoquinone-Induced Writhing Model

The PBQ-induced writhing model can be used to assess pain controlactivity of the peptides and GC-C receptor agonists of the invention.This model is described by Siegmund et al. (1957 Proc. Soc. Exp. Bio.Med. 95:729-731). Briefly, one hour after oral dosing with a testcompound, e.g., a peptide, morphine or vehicle, 0.02% phenylbenzoquinone(PBQ) solution (12.5 mL/kg) is injected by intraperitoneal route intothe mouse. The number of stretches and writhings are recorded from the5^(th) to the 10^(th) minute after PBQ injection, and can also becounted between the 35^(th) and 40^(th) minute and between the 60^(th)and 65^(th) minute to provide a kinetic assessment. The results areexpressed as the number of stretches and writhings (mean±SEM) and thepercentage of variation of the nociceptive threshold calculated from themean value of the vehicle-treated group. The statistical significance ofany differences between the treated groups and the control group isdetermined by a Dunnett's test using the residual variance after aone-way analysis of variance (P<0.05) using SigmaStat Software.

FIGS. 8 a and 8 b show the effect of different doses of MD-915 andMD-1100 in the PBQ writhing assay. Indomethacin, an NSAID (nonsteroidalanti-inflammatory drug) with known pain control activity, was used asthe positive control in the assay. Significant reductions in writhingswere observed for MD-915 (1 mg/kg dose) and MD-1100 (2.5 mg/kg dose)compared to the vehicle control. Loss of efficacy at the highest dosetested has also been observed for multiple other compounds (such as5HT-3 antagonists) tested in similar assays. The results of this studysuggest that both MD-915 and MD-1100 have antinociceptive effects inthis visceral pain model comparable to the intermediate doses ofindomethacin.

Example 5 MD-1100 Kd Determination

To determine the affinity of MD-1100 for GC-C receptors found in ratintestinal mucosa, a competition binding assay was performed using rateintestinal epithelial cells. Epithelial cells from the small intestineof rats were obtained as described by Kessler et al. (J. Biol. Chem.245: 5281-5288 (1970)). Briefly, animals were sacrificed and theirabdominal cavities exposed. The small intestine was rinsed with 300 mlice cold saline or PBS. 10 cm of the small intestine measured at 10 cmfrom the pylorus was removed and cut into 1 inch segments. Intestinalmucosa was extruded from the intestine by gentle pressure between apiece of parafilm and a P-1000 pipette tip. Intestinal epithelial cellswere placed in 2 ml PBS and pipetted up and down with a 5 ml pipette tomake a suspension of cells. Protein concentration in the suspension wasmeasured using the Bradford method (Anal. Biochem. 72: 248-254 (1976)).

A competition binding assay was performed based on the method ofGiannella et al. (Am. J. Physiol. 245: G492-G498) between [¹²⁵I] labeledMM-416776 and MD-1100. The assay mixture contained: 0.5 ml of DME with20 mM HEPES-KOH pH 7.0, 0.9 mg of the cell suspension listed above, 21.4fmol [¹²⁵I]-MM-416776 (42.8 pM), and different concentrations ofcompetitor MD-1100 (0.01 to 1000 nM). The mixture was incubated at roomtemperature for 1 hour, and the reaction stopped by applying the mixtureto GF/B glass-fiber filters (Whatman). The filters were washed with 5 mlice-cold PBS and radioactivity was measured. FIG. 9 shows that the Kdfor MD-1100 in this assay is 4.5 nm. % B/Bo is the percentage of theratio of radioactivity trapped in each sample (B) compared to theradioactivity retained in a control sample with no cold competitor (Bo).Giannella et al. (Am. J. Physiol. 245: G492-G498) observed that the Kdfor wild-type ST peptide in this same assay was ˜13 nm.

Example 6 Pharmacokinetic Properties of MD-1100

To study the pharmacokinetics of MD-1100, absorbability studies in micewere performed by administering MD-1100 intravenously via tail veininjection or orally by gavage to 8-week-old CD1 mice. Serum wascollected from the animals at various time points and tested for thepresence of MD-1100 using a competitive enzyme-linked immunoabsorbentassay (Oxoid, ST EIA kit, Cat#TD0700). The assay utilized monoclonalantibodies against ST peptide (antibodies are provided in the Oxoid kit)and synthetically manufactured MD-1100. FIG. 10 a shows absorption datafor intravenously and orally administered MD-1100 as detected by theELISA assay. MD-1100 appears to be minimally systemically absorbed andis <2.2% bioavailable.

A similar bioavailability study was performed in which LCMS rather thanELISA was used to detect MD-1100. Initially, serum samples wereextracted from the whole blood of exposed and control mice, theninjected directly (10 mL) onto an in-line solid phase extraction (SPE)column (Waters Oasis HLB 25 mm column, 2.0×15 mm direct connect) withoutfurther processing. The sample on the SPE column was washed with a 5%methanol, 95% dH₂O solution (2.1 mL/min, 1.0 minute), then loaded ontoan analytical column using a valve switch that places the SPE column inan inverted flow path onto the analytical column (Waters Xterra MS C8 5mm IS column, 2.1×20 mm). The sample was eluted from the analyticalcolumn with a reverse phase gradient (Mobile Phase A: 10 mM ammoniumhydroxide in dH₂O, Mobile Phase B: 10 mM ammonium hydroxide in 80%acetonitrile and 20% methanol; 20% B for the first 3 minutes thenramping to 95% B over 4 min. and holding for 2 min., all at a flow rateof 0.4 mL/min.). At 9.1 minutes, the gradient returns to the initialconditions of 20% B for 1 min. MD-1100 eluted from the analytical columnat 1.45 minutes, and was detected by triple-quadrapole mass spectrometry(MRM, 764 (+2 charge state)>182 (+1 charge state) Da; cone voltage=30V;collision=20 eV; parent resolution=2 Da at base peak; daughterresolution=2 Da at base peak). Instrument response was converted intoconcentration units by comparison with a standard curve using knownamounts of chemically synthesized MD-1100 prepared and injected in mouseserum using the same procedure.

FIG. 10 b shows absorption data for IV and orally administered MD-1100as detected by LCMS. In this assay, MD-1100 appears similarly minimallysystemically absorbed and is <0.11% bioavailable.

Administration of Peptides and GC-C Receptor Agonists

For treatment of gastrointestinal disorders, the peptides and agonistsof the invention are preferably administered orally, e.g., as a tabletor cachet containing a predetermined amount of the active ingredient,pellet, gel, paste, syrup, bolus, electuary, slurry, capsule; powder;granules; as a solution or a suspension in an aqueous liquid or anon-aqueous liquid; as an oil-in-water liquid emulsion or a water-in-oilliquid emulsion, via a liposomal formulation (see, e.g., EP 736299) orin some other form. Orally administered compositions can includebinders, lubricants, inert diluents, lubricating, surface active ordispersing agents, flavoring agents, and humectants. Orally administeredformulations such as tablets may optionally be coated or scored and maybe formulated so as to provide sustained, delayed or controlled releaseof the active ingredient therein. The peptides and agonists can beco-administered with other agents used to treat gastrointestinaldisorders including but not limited to acid suppressing agents such asHistamine-2 receptor agonists (H2As) and proton pump inhibitors (PPIs).The peptides and agonists can also be administered by rectalsuppository. For the treatment of disorders outside the gastrointestinaltract such as congestive heart failure and benign prostatic hypertrophy,peptides and agonists are preferably administered parenterally ororally.

The peptides described herein can be used alone or in combination withother agents. For example, the peptides can be administered togetherwith an analgesic peptide or compound. The analgesic peptide or compoundcan be covalently attached to a peptide described herein or it can be aseparate agent that is administered together with or sequentially with apeptide described herein in a combination therapy.

Combination therapy can be achieved by administering two or more agents,e.g., a peptide described herein and an analgesic peptide or compound,each of which is formulated and administered separately, or byadministering two or more agents in a single formulation. Othercombinations are also encompassed by combination therapy. For example,two agents can be formulated together and administered in conjunctionwith a separate formulation containing a third agent. While the two ormore agents in the combination therapy can be administeredsimultaneously, they need not be. For example, administration of a firstagent (or combination of agents) can precede administration of a secondagent (or combination of agents) by minutes, hours, days, or weeks.Thus, the two or more agents can be administered within minutes of eachother or within 1, 2, 3, 6, 9, 12, 15, 18, or 24 hours of each other orwithin 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 days of each other orwithin 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks of each other. In some caseseven longer intervals are possible. While in many cases it is desirablethat the two or more agents used in a combination therapy be present inwithin the patient's body at the same time, this need not be so.

Combination therapy can also include two or more administrations of oneor more of the agents used in the combination. For example, if agent Xand agent Y are used in a combination, one could administer themsequentially in any combination one or more times, e.g., in the orderX-Y-X, X-X-Y, Y-X-Y, Y-Y-X, X-X-Y-Y, etc.

The agents, alone or in combination, can be combined with anypharmaceutically acceptable carrier or medium. Thus, they can becombined with materials that do not produce an adverse, allergic orotherwise unwanted reaction when administered to a patient. The carriersor mediums used can include solvents, dispersants, coatings, absorptionpromoting agents, controlled release agents, and one or more inertexcipients (which include starches, polyols, granulating agents,microcrystalline cellulose, diluents, lubricants, binders,disintegrating agents, and the like), etc. If desired, tablet dosages ofthe disclosed compositions may be coated by standard aqueous ornonaqueous techniques.

Compositions of the present invention may also optionally include othertherapeutic ingredients, anti-caking agents, preservatives, sweeteningagents, colorants, flavors, desiccants, plasticizers, dyes, and thelike. Any such optional ingredient must be compatible with the compoundof the invention to insure the stability of the formulation. Thecomposition may contain other additives as needed, including for examplelactose, glucose, fructose, galactose, trehalose, sucrose, maltose,raffinose, maltitol, melezitose, stachyose, lactitol, palatinite,starch, xylitol, mannitol, myoinositol, and the like, and hydratesthereof, and amino acids, for example alanine, glycine and betaine, andpeptides and proteins, for example albumen.

Examples of excipients for use as the pharmaceutically acceptablecarriers and the pharmaceutically acceptable inert carriers and theaforementioned additional ingredients include, but are not limited tobinders, fillers, disintegrants, lubricants, anti-microbial agents, andcoating agents such as:

BINDERS: corn starch, potato starch, other starches, gelatin, naturaland synthetic gums such as acacia, sodium alginate, alginic acid, otheralginates, powdered tragacanth, guar gum, cellulose and its derivatives(e.g., ethyl cellulose, cellulose acetate, carboxymethyl cellulosecalcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methylcellulose, pre-gelatinized starch (e.g., STARCH 1500® and STARCH 1500LM®, sold by Colorcon, Ltd.), hydroxypropyl methyl cellulose,microcrystalline cellulose (e.g. AVICEL™, such as, AVICEL-PH-101™, -103™and -105™, sold by FMC Corporation, Marcus Hook, Pa., USA), or mixturesthereof,

FILLERS: talc, calcium carbonate (e.g., granules or powder), dibasiccalcium phosphate, tribasic calcium phosphate, calcium sulfate (e.g.,granules or powder), microcrystalline cellulose, powdered cellulose,dextrates, kaolin, mannitol, silicic acid, sorbitol, starch,pre-gelatinized starch, or mixtures thereof,

DISINTEGRANTS: agar-agar, alginic acid, calcium carbonate,microcrystalline cellulose, croscarmellose sodium, crospovidone,polacrilin potassium, sodium starch glycolate, potato or tapioca starch,other starches, pre-gelatinized starch, clays, other algins, othercelluloses, gums, or mixtures thereof,

LUBRICANTS: calcium stearate, magnesium stearate, mineral oil, lightmineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, otherglycols, stearic acid, sodium lauryl sulfate, talc, hydrogenatedvegetable oil (e.g., peanut oil, cottonseed oil, sunflower oil, sesameoil, olive oil, corn oil and soybean oil), zinc stearate, ethyl oleate,ethyl laurate, agar, syloid silica gel (AEROSIL 200, W.R. Grace Co.,Baltimore, Md. USA), a coagulated aerosol of synthetic silica (DeaussaCo., Plano, Tex. USA), a pyrogenic silicon dioxide (CAB-O-SIL, CabotCo., Boston, Mass. USA), or mixtures thereof,

ANTI-CAKING AGENTS: calcium silicate, magnesium silicate, silicondioxide, colloidal silicon dioxide, talc, or mixtures thereof,

ANTIMICROBIAL AGENTS: benzalkonium chloride, benzethonium chloride,benzoic acid, benzyl alcohol, butyl paraben, cetylpyridinium chloride,cresol, chlorobutanol, dehydroacetic acid, ethylparaben, methylparaben,phenol, phenylethyl alcohol, phenoxyethanol, phenylmercuric acetate,phenylmercuric nitrate, potassium sorbate, propylparaben, sodiumbenzoate, sodium dehydroacetate, sodium propionate, sorbic acid,thimersol, thymo, or mixtures thereof, and

COATING AGENTS: sodium carboxymethyl cellulose, cellulose acetatephthalate, ethylcellulose, gelatin, pharmaceutical glaze, hydroxypropylcellulose, hydroxypropyl methylcellulose, hydroxypropyl methyl cellulosephthalate, methylcellulose, polyethylene glycol, polyvinyl acetatephthalate, shellac, sucrose, titanium dioxide, carnauba wax,microcrystalline wax, or mixtures thereof.

The agents either in their free form or as a salt can be combined with apolymer such as polylactic-glycolic acid (PLGA),poly-(I)-lactic-glycolic-tartaric acid (P(I)LGT) (WO 01/12233),polyglycolic acid (U.S. Pat. No. 3,773,919), polylactic acid (U.S. Pat.No. 4,767,628), poly(M-caprolactone) and poly(alkylene oxide) (U.S.20030068384) to create a sustained release formulation. Suchformulations can be used to implants that release a peptide or anotheragent over a period of a few days, a few weeks or several monthsdepending on the polymer, the particle size of the polymer, and the sizeof the implant (see, e.g., U.S. Pat. No. 6,620,422). Other sustainedrelease formulations are described in EP 0 467 389 A2, WO 93/241150,U.S. Pat. No. 5,612,052, WO 97/40085, WO 03/075887, WO 01/01964A2, U.S.Pat. No. 5,922,356, WO 94/155587, WO 02/074247A2, WO 98/25642, U.S. Pat.No. 5,968,895, U.S. Pat. No. 6,180,608, U.S. 20030171296, U.S.20020176841, U.S. Pat. No. 5,672,659, U.S. Pat. No. 5,893,985, U.S. Pat.No. 5,134,122, U.S. Pat. No. 5,192,741, U.S. Pat. No. 5,192,741, U.S.Pat. No. 4,668,506, U.S. Pat. No. 4,713,244, U.S. Pat. No. 5,445,832U.S. Pat. No. 4,931,279, U.S. Pat. No. 5,980,945, WO 02/058672, WO9726015, WO 97/04744, and. US20020019446. In such sustained releaseformulations microparticles of peptide are combined with microparticlesof polymer. One or more sustained release implants can be placed in thelarge intestine, the small intestine or both. U.S. Pat. No. 6,011,011and WO 94/06452 describe a sustained release formulation providingeither polyethylene glycols (where PEG 300 and PEG 400 are mostpreferred) or triacetin. WO 03/053401 describes a formulation which mayboth enhance bioavailability and provide controlled release of the agentwithin the GI tract. Additional controlled release formulations aredescribed in WO 02/38129, EP 326 151, U.S. Pat. No. 5,236,704, WO02/30398, WO 98/13029; U.S. 20030064105, U.S. 20030138488A1, U.S.20030216307A1,U.S. Pat. No. 6,667,060, WO 01/49249, WO 01/49311, WO01/49249, WO 01/49311, and U.S. Pat. No. 5,877,224.

The agents can be administered, e.g., by intravenous injection,intramuscular injection, subcutaneous injection, intraperitonealinjection, topical, sublingual, intraarticular (in the joints),intradermal, buccal, ophthalmic (including intraocular), intranasally(including using a cannula), or by other routes. The agents can beadministered orally, e.g., as a tablet or cachet containing apredetermined amount of the active ingredient, gel, pellet, paste,syrup, bolus, electuary, slurry, capsule, powder, granules, as asolution or a suspension in an aqueous liquid or a non-aqueous liquid,as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion,via a micellar formulation (see, e.g. WO 97/11682) via a liposomalformulation (see, e.g., EP 736299, WO 99/59550 and WO 97/13500), viaformulations described in WO 03/094886 or in some other form. Orallyadministered compositions can include binders, lubricants, inertdiluents, lubricating, surface active or dispersing agents, flavoringagents, and humectants. Orally administered formulations such as tabletsmay optionally be coated or scored and may be formulated so as toprovide sustained, delayed or controlled release of the activeingredient therein. The agents can also be administered transdermally(i.e. via reservoir-type or matrix-type patches, microneedles, thermalporation, hypodermic needles, iontophoresis, electroporation, ultrasoundor other forms of sonophoresis, jet injection, or a combination of anyof the preceding methods (Prausnitz et al. 2004, Nature Reviews DrugDiscovery 3:115-124)). The agents can be administered usinghigh-velocity transdermal particle injection techniques using thehydrogel particle formulation described in U.S. 20020061336. Additionalparticle formulations are described in WO 00/45792, WO 00/53160, and WO02/19989. An example of a transdermal formulation containing plaster andthe absorption promoter dimethylisosorbide can be found in WO 89/04179.WO 96/11705 provides formulations suitable for transdermaladministration. The agents can be administered in the form a suppositoryor by other vaginal or rectal means. The agents can be administered in atransmembrane formulation as described in WO 90/07923. The agents can beadministered non-invasively via the dehydrated particles described inU.S. Pat. No. 6,485,706. The agent can be administered in anenteric-coated drug formulation as described in WO 02/49621. The agentscan be administered intranassaly using the formulation described in U.S.Pat. No. 5,179,079. Formulations suitable for parenteral injection aredescribed in WO 00/62759. The agents can be administered using thecasein formulation described in U.S. 20030206939 and WO 00/06108. Theagents can be administered using the particulate formulations describedin U.S. 20020034536.

The agents, alone or in combination with other suitable components, canbe administered by pulmonary route utilizing several techniquesincluding but not limited to intratracheal instillation (delivery ofsolution into the lungs by syringe), intratracheal delivery ofliposomes, insufflation (administration of powder formulation by syringeor any other similar device into the lungs) and aerosol inhalation.Aerosols (e.g., jet or ultrasonic nebulizers, metered-dose inhalers(MDIs), and dry-powder inhalers (DPIs)) can also be used in intranasalapplications. Aerosol formulations are stable dispersions or suspensionsof solid material and liquid droplets in a gaseous medium and can beplaced into pressurized acceptable propellants, such ashydrofluoroalkanes (HFAs, i.e. HFA-134a and HFA-227, or a mixturethereof), dichlorodifluoromethane (or other chlorofluocarbon propellantssuch as a mixture of Propellants 11, 12, and/or 114), propane, nitrogen,and the like. Pulmonary formulations may include permeation enhancerssuch as fatty acids, and saccharides, chelating agents, enzymeinhibitors (e.g., protease inhibitors), adjuvants (e.g., glycocholate,surfactin, span 85, and nafamostat), preservatives (e.g., benzalkoniumchloride or chlorobutanol), and ethanol (normally up to 5% but possiblyup to 20%, by weight). Ethanol is commonly included in aerosolcompositions as it can improve the function of the metering valve and insome cases also improve the stability of the dispersion. Pulmonaryformulations may also include surfactants which include but are notlimited to bile salts and those described in U.S. Pat. No. 6,524,557 andreferences therein. The surfactants described in U.S. Pat. No.6,524,557, e.g., a C8-C16 fatty acid salt, a bile salt, a phospholipid,or alkyl saccharide are advantageous in that some of them alsoreportedly enhance absorption of the peptide in the formulation. Alsosuitable in the invention are dry powder formulations comprising atherapeutically effective amount of active compound blended with anappropriate carrier and adapted for use in connection with a dry-powderinhaler. Absorption enhancers which can be added to dry powderformulations of the present invention include those described in U.S.Pat. No. 6,632,456. WO 02/080884 describes new methods for the surfacemodification of powders. Aerosol formulations may include U.S. Pat. No.5,230,884, U.S. Pat. No. 5,292,499, WO 017/8694, WO 01/78696, U.S.2003019437, U.S. 20030165436, and WO 96/40089 (which includes vegetableoil). Sustained release formulations suitable for inhalation aredescribed in U.S. 20010036481A1, 20030232019A1, and U.S. 20040018243A1as well as in WO 01/13891, WO 02/067902, WO 03/072080, and WO 03/079885.Pulmonary formulations containing microparticles are described in WO03/015750, U.S. 20030008013, and WO 00/00176. Pulmonary formulationscontaining stable glassy state powder are described in U.S. 20020141945and U.S. Pat. No. 6,309,671. Other aerosol formulations are described inEP 1338272A1 WO 90/09781, U.S. Pat. No. 5,348,730, U.S. Pat. No.6,436,367, WO 91/04011, and U.S. Pat. No. 6,294,153 and U.S. Pat. No.6,290,987 describes a liposomal based formulation that can beadministered via aerosol or other means. Powder formulations forinhalation are described in U.S. 20030053960 and WO 01/60341. The agentscan be administered intranasally as described in U.S. 20010038824.

Solutions of medicament in buffered saline and similar vehicles arecommonly employed to generate an aerosol in a nebulizer. Simplenebulizers operate on Bernoulli's principle and employ a stream of airor oxygen to generate the spray particles. More complex nebulizersemploy ultrasound to create the spray particles. Both types are wellknown in the art and are described in standard textbooks of pharmacysuch as Sprowls'American Pharmacy and Remington's The Science andPractice of Pharmacy. Other devices for generating aerosols employcompressed gases, usually hydrofluorocarbons and chlorofluorocarbons,which are mixed with the medicament and any necessary excipients in apressurized container, these devices are likewise described in standardtextbooks such as Sprowls and Remington.

The agents can be a free acid or base, or a pharmacologically acceptablesalt thereof. Solids can be dissolved or dispersed immediately prior toadministration or earlier. In some circumstances the preparationsinclude a preservative to prevent the growth of microorganisms. Thepharmaceutical forms suitable for injection can include sterile aqueousor organic solutions or dispersions which include, e.g., water, analcohol, an organic solvent, an oil or other solvent or dispersant(e.g., glycerol, propylene glycol, polyethylene glycol, and vegetableoils). The formulations may contain antioxidants, buffers,bacteriostats, and solutes that render the formulation isotonic with theblood of the intended recipient, and aqueous and non-aqueous sterilesuspensions that can include suspending agents, solubilizers, thickeningagents, stabilizers, and preservatives. Pharmaceutical agents can besterilized by filter sterilization or by other suitable means.

The agent can be fused to immunoglobulins or albumin, or incorporatedinto a liposome to improve half-life. The agent can also be conjugatedto polyethylene glycol (PEG) chains. Methods for pegylation andadditional formulations containing PEG-conjugates (i.e. PEG-basedhydrogels, PEG modified liposomes) can be found in Harris and Chess,Nature Reviews Drug Discovery 2: 214-221 and the references therein. Theagent can be administered via a nanocochleate or cochleate deliveryvehicle (BioDelivery Sciences International). The agents can bedelivered transmucosally (i.e. across a mucosal surface such as thevagina, eye or nose) using formulations such as that described in U.S.Pat. No. 5,204,108. The agents can be formulated in microcapsules asdescribed in WO 88/01165. The agent can be administered intra-orallyusing the formulations described in U.S. 20020055496, WO 00/47203, andU.S. Pat. No. 6,495,120. The agent can be delivered using nanoemulsionformulations described in WO 01/91728A2.

Suitable pharmaceutical compositions in accordance with the inventionwill generally include an amount of the active compound(s) with anacceptable pharmaceutical diluent or excipient, such as a sterileaqueous solution, to give a range of final concentrations, depending onthe intended use. The techniques of preparation are generally well knownin the art, as exemplified by Remington's Pharmaceutical Sciences (18thEdition, Mack Publishing Company, 1995).

The agents described herein and combination therapy agents can bepackaged as a kit that includes single or multiple doses of two or moreagents, each packaged or formulated individually, or single or multipledoses of two or more agents packaged or formulated in combination. Thus,one or more agents can be present in first container, and the kit canoptionally include one or more agents in a second container. Thecontainer or containers are placed within a package, and the package canoptionally include administration or dosage instructions. A kit caninclude additional components such as syringes or other means foradministering the agents as well as diluents or other means forformulation.

Methods to increase chemical and/or physical stability of the agents thedescribed herein are found in U.S. Pat. No. 6,541,606, U.S. Pat. No.6,068,850, U.S. Pat. No. 6,124,261, U.S. Pat. No. 5,904,935, and WO00/15224, U.S. 20030069182 (via the addition of nicotinamide), U.S.20030175230A1, U.S. 20030175230A1, U.S. 20030175239A1, U.S. 20020045582,U.S. 20010031726, WO 02/26248, WO 03/014304, WO 98/00152A1, WO98/00157A1, WO 90/12029, WO 00/04880, and WO 91/04743, WO 97/04796 andthe references cited therein.

Methods to increase bioavailability of the agents described herein arefound in U.S. Pat. No. 6,008,187, U.S. Pat. No. 5,424,289, U.S.20030198619, WO 90/01329, WO 01/49268, WO 00/32172, and WO 02/064166.Glycyrrhizinate can also be used as an absorption enhancer (see, e.g.,EP397447). WO 03/004062 discusses Ulex europaeus I (UEAI) and UEAImimetics which may be used to target the agents of the invention to theGI tract.

Analgesic Agents

The peptides described herein can be used in combination therapy with ananalgesic agent, e.g., an analgesic compound or an analgesic peptide.The analgesic agent can optionally be covalently attached to a peptidedescribed herein. Among the useful analgesic agents are: Ca channelblockers, 5HT receptor antagonists (for example 5HT3, 5HT4 and 5HT1receptor antagonists), opioid receptor agonists (loperamide, fedotozine,and fentanyl), NK1 receptor antagonists, CCK receptor agonists (e.g.,loxiglumide), NK1 receptor antagonists, NK3 receptor antagonists,norepinephrine-serotonin reuptake inhibitors (NSRI), vanilloid andcannabanoid receptor agonists, and sialorphin. Analgesics agents in thevarious classes are described in the literature.

Among the useful analgesic peptides are sialorphin-related peptides,including those comprising the amino acid sequence QHNPR (SEQ IDNO:111), including: VQHNPR (SEQ ID NO:112); VRQHNPR (SEQ ID NO:113);VRGQHNPR (SEQ ID NO:114); VRGPQHNPR (SEQ ID NO:115); VRGPRQHNPR (SEQ IDNO:116); VRGPRRQHNPR (SEQ ID NO:117); and RQHNPR (SEQ ID NO:118).Sialorphin-related peptides bind to neprilysin and inhibitneprilysin-mediated breakdown of substance P and Met-enkephalin. Thus,compounds or peptides that are inhibitors of neprilysin are usefulanalgesic agents which can be administered with the peptides of theinvention in a co-therapy or linked to the peptides of the invention,e.g., by a covalent bond. Sialophin and related peptides are describedin U.S. Pat. No. 6,589,750; U.S. 20030078200 A1; and WO 02/051435 A2.

Opioid receptor antagonists and agonists can be administered with thepeptides of the invention in co-therapy or linked to the peptide of theinvention, e.g., by a covalent bond. For example, opioid receptorantagonists such as naloxone, naltrexone, methyl nalozone, nalmefene,cypridime, beta funaltrexamine, naloxonazine, naltrindole, andnor-binaltorphimine are thought to be useful in the treatment of IBS. Itcan be useful to formulate opioid antagonists of this type is a delayedand sustained release formulation such that initial release of theantagonist is in the mid to distal small intestine and/or ascendingcolon. Such antagonists are described in WO 01/32180 A2. Enkephalinpentapeptide (HOE825; Tyr-D-Lys-Gly-Phe-L-homoserine) is an agonist ofthe mu and delta opioid receptors and is thought to be useful forincreasing intestinal motility (Eur. J. Pharm. 219:445, 1992), and thispeptide can be used in conjunction with the peptides of the invention.Also useful is trimebutine which is thought to bind to mu/delta/kappaopioid receptors and activate release of motilin and modulate therelease of gastrin, vasoactive intestinal peptide, gastrin andglucagons. Kappa opioid receptor agonists such as fedotozine,ketocyclazocine, and compounds described in WO 03/097051 A2 can be usedwith or linked to the peptides of the invention. In addition, mu opioidreceptor agonists such as morphine, diphenyloxylate, frakefamide(H-Tyr-D-Ala-Phe(F)-Phe-NH₂; WO 01/019849 A1) and loperamide can beused.

Tyr-Arg (kyotorphin) is a dipeptide that acts by stimulating the releaseof met-enkephalins to elicit an analgesic effect (J. Biol. Chem.262:8165, 1987). Kyotorphin can be used with or linked to the peptidesof the invention.

CCK receptor agonists such as caerulein from amphibians and otherspecies are useful analgesic agents that can be used with or linked tothe peptides of the invention.

Conotoxin peptides represent a large class of analgesic peptides thatact at voltage gated Ca channels, NMDA receptors or nicotinic receptors.These peptides can be used with or linked to the peptides of theinvention.

Peptide analogs of thymulin (FR 2830451) can have analgesic activity andcan be used with or linked to the peptides of the invention.

CCK (CCKa or CCKb) receptor antagonists, including loxiglumide anddexloxiglumide (the R-isomer of loxiglumide) (WO 88/05774) can haveanalgesic activity and can be used with or linked to the peptides of theinvention.

Other useful analgesic agents include 5-HT4 agonists such astegaserod/zelnorm and lirexapride. Such agonists are described in:EP1321142 A1, WO 03/053432A1, EP 505322 A1, EP 505322 B1, U.S. Pat. No.5,510,353, EP 507672 A1, EP 507672 B1, and U.S. Pat. No. 5,273,983.

Calcium channel blockers such as ziconotide and related compoundsdescribed in, for example, EP 625162B1, U.S. Pat. No. 5,364,842, U.S.Pat. No. 5,587,454, U.S. Pat. No. 5,824,645, U.S. Pat. No. 5,859,186,U.S. Pat. No. 5,994,305, U.S. Pat. No. 6,087,091, U.S. Pat. No.6,136,786, WO 93/13128 A1, EP 1336409 A1, EP 835126 A1, EP 835126 B1,U.S. Pat. No. 5,795,864, U.S. Pat. No. 5,891,849, U.S. Pat. No.6,054,429, WO 97/01351 A1, can be used with or linked to the peptides ofthe invention.

Various antagonists of the NK-1, NK-2, and NK-3 receptors (for a reviewsee Giardina et al. 2003 Drugs 6:758) can be can be used with or linkedto the peptides of the invention.

NK1 receptor antagonists such as: aprepitant (Merck & Co Inc),vofopitant, ezlopitant (Pfizer, Inc.), R-673 (Hoffmann-La Roche Ltd),SR-14033 and related compounds described in, for example, EP 873753 A1,U.S. 20010006972 A1, U.S. 20030109417 A1, WO 01/52844 A1, can be usedwith or linked to the peptides of the invention.

NK-2 receptor antagonists such as nepadutant (Menarini Ricerche SpA),saredutant (Sanofi-Synthelabo), SR-144190 (Sanofi-Synthelabo) andUK-290795 (Pfizer Inc) can be used with or linked to the peptides of theinvention.

NK3 receptor antagonists such as osanetant (Sanofi-Synthelabo),talnetant and related compounds described in, for example, WO 02/094187A2, EP 876347 A1, WO 97/21680 A1, U.S. Pat. No. 6,277,862, WO 98/11090,WO 95/28418, WO 97/19927, and Boden et al. (J Med Chem. 39:1664-75,1996) can be used with or linked to the peptides of the invention.

Norepinephrine-serotonin reuptake inhibitors such as milnacipran andrelated compounds described in WO 03/077897 A1 can be used with orlinked to the peptides of the invention.

Vanilloid receptor antagonists such as arvanil and related compoundsdescribed in WO 01/64212 A1 can be used with or linked to the peptidesof the invention.

Where the analgesic is a peptide and is covalently linked to a peptidedescribed herein the resulting peptide may also include at least onetrypsin or chymotrypsin cleavage site. When present within the peptide,the analgesic peptide may be preceded by (if it is at the carboxyterminus) or followed by (if it is at the amino terminus) a chymotrypsinor trypsin cleavage site that allows release of the analgesic peptide.

In addition to sialorphin-related peptides, analgesic peptides include:AspPhe, endomorphin-1, endomorphin-2, nocistatin, dalargin, lupron,zicnotide, and substance P.

Methods of Treatment

The peptides of the invention can be used for the treatment orprevention of cancer, pre-cancerous growths, or metastatic growths. Forexample, they can be used for the prevention or treatment of:colorectal/local metastasized colorectal cancer, gastrointestinal tractcancer, lung cancer, cancer or pre-cancerous growths or metastaticgrowths of epithelial cells, polyps, breast, colorectal, lung, ovarian,pancreatic, prostatic, renal, stomach, bladder, liver, esophageal andtesticular carcinoma, carcinoma (e.g., basal cell, basosquamous,Brown-Pearce, ductal carcinoma, Ehrlich tumor, Krebs, Merkel cell, smallor non-small cell lung, oat cell, papillary, bronchiolar, squamous cell,transitional cell, Walker), leukemia (e.g., B-cell, T-cell, HTLV, acuteor chronic lymphocytic, mast cell, myeloid), histiocytonia,histiocytosis, Hodgkin's disease, non-Hodgkin's lymphoma, plasmacytoma,reticuloendotheliosis, adenoma, adeno-carcinoma, adenofibroma,adenolymphoma, ameloblastoma, angiokeratoma, angiolymphoid hyperplasiawith eosinophilia, sclerosing angioma, angiomatosis, apudoma,branchionia, malignant carcinoid syndrome, carcinoid heart disease,carcinosarcoma, cementoma, cholangioma, cholesteatoma, chondrosarcoma,chondroblastoma, chondrosarcoma, chordoma, choristoma,craniopharyngioma, chrondrorna, cylindroma, cystadenocarcinoma,cystadenoma, cystosarconia phyllodes, dysgenninoma, ependymoma, Ewingsarcoma, fibroma, fibrosarcoma, giant cell tumor, ganglioneuroma,glioblastoma, glomangioma, granulosa cell tumor, gynandroblastoma,hamartoma, hemangioendothelioma, hemangioma, hemangio-pericytoma,hemangiosarcoma, hepatoma, islet cell tumor, Kaposi sarcoma, leiomyoma,leiomyosarcoma, leukosarcoma, Leydig cell tumor, lipoma, liposarcoma,lymphaugioma, lymphangiomyoma, lymphangiosarcoma, medulloblastoma,meningioma, mesenchymoma, mesonephroma, mesothelioma, myoblastoma,myoma, myosarcoma, myxoma, myxosarcoma, neurilemmoma, neuroma,neuroblastoma, neuroepithelioma, neurofibroma, neurofibromatosis,odontoma, osteoma, osteosarcoma, papilloma, paraganglioma,paraganglionia. nonchroinaffin, pinealoma, rhabdomyoma,rhabdomyosarcoma, Sertoli cell tumor, teratoma, theca cell tumor, andother diseases in which cells have become dysplastic, immortalized, ortransformed.

The peptides of the invention can be used for the treatment orprevention of: Familial Adenomatous Polyposis (FAP) (autosomal dominantsyndrome) that precedes colon cancer, hereditary nonpolyposis colorectalcancer (HNPCC), and inherited autosomal dominant syndrome.

For treatment or prevention of cancer, pre-cancerous growths andmetastatic growths, the peptides can be used in combination therapy withradiation or chemotherapeutic agents, an inhibitor of a cGMP-dependentphosphodiesterase or a selective cyclooxygenase-2 inhibitor (a number ofselective cyclooxygenase-2 inhibitors are described in WO02062369,hereby incorporated by reference).

The peptides can be for treatment or prevention of inflammation. Thus,they can be used alone or in combination with inhibitor ofcGMP-dependent phosphodiesterase or a selective cyclooxygenase-2inhibitor for treatment of: organ inflammation, IBD (e.g., Crohn'sdisease, ulcerative colitis), asthma, nephritis, hepatitis,pancreatitis, bronchitis, cystic fibrosis, ischemic bowel diseases,intestinal inflammations/allergies, coeliac disease, proctitis,eosnophilic gastroenteritis, mastocytosis, and other inflammatorydisorders.

The peptides can also be used to treat or prevent insulin-relateddisorders, for example: II diabetes mellitus, hyperglycemia, obesity,disorders associated with disturbances in glucose or electrolytetransport and insulin secretion in cells, or endocrine disorders. Theycan be also used in insulin resistance treatment and post-surgical andnon-post surgery decrease in insulin responsiveness.

The peptides can be used to prevent or treat respiratory disorders,including, inhalation, ventilation and mucus secretion disorders,pulmonary hypertension, chronic obstruction of vessels and airways, andirreversible obstructions of vessels and bronchi.

The peptides can be used in combination therapy with a phosphodiesteraseinhibitor (examples of such inhibitors can be found in U.S. Pat. No.6,333,354, hereby incorporated by reference).

The peptides can also be used to prevent or treat: retinopathy,nephropathy, diabetic angiopathy, and edema formation

The peptides can also be used to prevent or treat neurologicaldisorders, for example, headache, anxiety, movement disorders,aggression, psychosis, seizures, panic attacks, hysteria, sleepdisorders, depression, schizoaffective disorders, sleep apnea, attentiondeficit syndromes, memory loss, and narcolepsy. They may also be used asa sedative.

The peptides and detectably labeled peptides can be used as markers toidentify, detect, stage, or diagnosis diseases and conditions of thesmall intestine, including: Crohn's disease, colitis, inflammatory boweldisease, tumors, benign tumors, such as benign stromal tumors, adenoma,angioma, adenomatous (pedunculated and sessile) polyps, malignant,carcinoid tumors, endocrine cell tumors, lymphoma, adenocarcinoma,foregut, midgut, and hindgut carcinoma, gastroinstestinal stromal tumor(GIST), such as leiomyoma, cellular leiomyoma, leiomyoblastoma, andleiomyosarcoma, gastrointestinal autonomic nerve tumor, malabsorptionsyndromes, celiac diseases, diverticulosis, Meckel's diverticulum,colonic diverticula, megacolon, Hirschsprung's disease, irritable bowelsyndrome, mesenteric ischemia, ischemic colitis, colorectal cancer,colonic polyposis, polyp syndrome, intestinal adenocarcinoma, Liddlesyndrome, Brody myopathy, infantile convulsions, and choreoathetosis

The peptides can be conjugated to another molecule (e.g., a diagnosticor therapeutic molecule) to target cells bearing the GCC receptor, e.g.,cystic fibrosis lesions and specific cells lining the intestinal tract.Thus, they can be used to target radioactive moieties or therapeuticmoieties to the intestine to aid in imaging and diagnosing or treatingcolorectal/metastasized or local colorectal cancer and to deliver normalcopies of the p53 tumor suppressor gene to the intestinal tract.

The peptides can be used alone or in combination therapy to treaterectile dysfunction.

The peptides can be used alone or in combination therapy to treat innerear disorders, e.g., to treat Meniere's disease, including symptoms ofthe disease such as vertigo, hearing loss, tinnitus, sensation offullness in the ear, and to maintain fluid homeostasis in the inner ear.

The peptides can be used alone or in combination therapy to treatdisorders associated with fluid and sodium retention, e.g., diseases ofthe electrolyte-water/electrolyte transport system within the kidney,gut and urogenital system, congestive heart failure, hypertension,hypotension, liver cirrhosis, and nephrotic syndrome. In addition theycan be used to facilitate diuresis or control intestinal fluid.

The peptides can be used alone or in combination therapy to treatdisorders associated with chloride or bicarbonate secretion, e.g.,Cystic Fibrosis.

The peptides can be used alone or in combination therapy to treatdisorders associated with bile secretion. In addition, they can be usedto facilitate or control chloride and bile fluid secretion in the gallbladder.

The peptides can be used alone or in combination therapy to treatdisorders associated with liver cell regeneration.

1-19. (canceled)
 20. A method for increasing intestinal motility in apatient, the method comprising administering to the patient an effectiveamount of a peptide comprising the amino acid sequence Cys Cys Glu TyrCys Cys Asn Pro Ala Cys Thr Gly Cys (SEQ ID NO:125) or apharmaceutically acceptable salt thereof.
 21. The method of claim 20,wherein the peptide consists of the amino acid sequence Cys Cys Glu TyrCys Cys Asn Pro Ala Cys Thr Gly Cys (SEQ ID NO:125) or apharmaceutically acceptable salt thereof.
 22. A method for treating apatient for a gastrointestinal disorder, the method comprisingadministering to the patient an effective amount of a peptide comprisingthe amino acid sequence Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr GlyCys (SEQ ID NO:125) or a pharmaceutically acceptable salt thereof. 23.The method of claim 22, wherein the peptide consists of the amino acidsequence Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys (SEQ IDNO:125) or a pharmaceutically acceptable salt thereof.
 24. The method ofclaim 22, wherein the gastrointestinal disorder is selected from thegroup consisting of: irritable bowel syndrome, constipation, agastrointestinal motility disorder, gastroesophageal reflux disease,functional heartburn, dyspepsia, gastroparesis, chronic intestinalpseudo-obstruction and colonic pseudo-obstruction.
 25. The method ofclaim 24, wherein the gastrointestinal disorder is constipation.
 26. Themethod of claim 25, wherein the constipation is chronic constipation,idiopathic constipation, constipation due to post-operative ileus, orconstipation caused by opiate use.
 27. The method of claim 26, whereinthe constipation is chronic constipation.
 28. The method of claim 24,wherein the gastrointestinal disorder is irritable bowel syndrome. 29.The method of claim 28, wherein the irritable bowel syndrome isconstipation-predominant irritable bowel syndrome.
 30. The method ofclaim 28, wherein the irritable bowel syndrome is alternating irritablebowel syndrome.
 31. A method for preparing a peptide comprising theamino acid sequence Cys Cys Glu Tyr Cys Cys Asn Pro Ala Cys Thr Gly Cys(SEQ ID NO:125) or a pharmaceutically acceptable salt thereof, themethod comprising: (a) chemically synthesizing the peptide; and (b)purifying the peptide.
 32. The method of claim 31, wherein the methodfurther comprises chemically synthesizing the peptide with solid phasechemical synthesis using a double coupling program and protected thiol,carboxyl, and hydroxyl groups of the peptide.
 33. The method of claim32, wherein the method further comprises purifying the peptide withreverse phase chromatography.